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Background: Antiphospholipid antibody (aPL) testing is critical for the classification of antiphospholipid syndrome. The 2023 ACR/EULAR classification criteria recommend the use of enzyme-linked immunosorbent assays (ELISAs) and specific thresholds for aPL positivity. Since non-ELISA methods are increasingly used, we compared and evaluated ELISA and non-ELISA aPL assays in a real-world maximum care hospital setting. Methods: Between January 2021 and June 2024, anticardiolipin (aCL; IgG and IgM) and anti-beta2 glycoprotein I (aß2GPI; IgG and IgM) antibodies were measured using ELISA (n = 5115) and a chemiluminescence-based automated immunoassay (CLIA) (n = 3820). Results of parallel testing were compared, and associations with clinical and laboratory characteristics were evaluated. Results: A total of 946 samples were tested using ELISA and CLIA in parallel. A total of 136 (14%) specimens were positive for at least one aPL, and 55 (6%) specimens were from patients diagnosed with APS. Among the latter, 47 (85%) and 41 (75%) patients were positive when ELISA- or CLIA-based aPL assays were used, respectively. After applying the >40 units threshold of the new classification criteria, the number of aPL-positive specimens was significantly lower. In the entire cohort, the agreement between ELISA and CLIA aPL assays was acceptable only for aß2GPI IgG; the results from the two methods did not agree for aCL IgG/IgM and aß2GPI IgM. In APS patients, the agreement between ELISA and CLIA aPL assays was acceptable for aß2GPI IgG and IgM but poor for aCL IgG and IgM. Antibody levels in APS patients were significantly higher using CLIA compared to ELISA. Conclusions: The method-dependent discrepancies between ELISA- and CLIA-based aPL assays regarding the quantitative and qualitative results are substantial. Both methods are suitable for APS classification, but the choice of aPL assay may influence the classification, and therefore, aPL results should be interpreted carefully in the clinical context. [ABSTRACT FROM AUTHOR]

There was evidence that perinuclear antineutrophil cytoplasmic antibodies (pANCA) in autoimmune liver diseases react with human beta-tubulin-5 (TBB5). Here, we reevaluate the specificity and clinical relevance of anti-TBB5 antibodies. Patients with untreated autoimmune hepatitis (AIH; n = 53), AIH under immunosuppressive therapy (AIH-IS; n = 125), primary sclerosing cholangitis (PSC; n = 40), primary biliary cholangitis (PBC; n = 250), nonautoimmune liver diseases (n = 158), inflammatory bowel diseases (IBD; n = 30), and healthy individuals (n = 62) were tested by enzyme-linked immunosorbent assay for IgG- and IgA-antibodies against recombinant human TBB5. pANCA were detected by immunofluorescence test. Sera were absorbed with TBB5 coupled to cyanogen bromide-activated sepharose. Prevalence and reactivity of IgG anti-TBB5 were significantly higher in patients with untreated AIH (68%; arbitrary units [AU] median: 369) than in PSC (28%; AU median: 84, P < 0.001), other liver diseases (14%; AU median: 185, P < 0.0001), IBD (3%; AU median: 111, P < 0.0001), and healthy controls (3%; AU median: 135; P < 0.0001). Anti-TBB5 did not correlate with pANCA, and immunoprecipitation with TBB5 did not abolish pANCA reactivity. In untreated AIH, anti-TBB5-reactivity was significantly higher than in AIH-IS. Transaminases decreased under IS preferentially in anti-TBB5-negative patients. There was no correlation between anti-TBB5-reactivity and histological stages. IgA-anti-TBB5 was mainly found in alcohol-associated liver disease (ALD; 39%). Our data do not support TBB5 as an autoantigenic target of pANCA. However, IgG-anti-TBB5 showed high specificity for (untreated) AIH. While they did not correlate with histological and laboratory parameters, their presence may indicate a poor response to IS. Sera from untreated AIH patients show significantly higher reactivity of IgG anti-TBB5 antibodies than sera from patients with other liver disorders and controls in the enzyme-linked immunosorbent assay using recombinant TBB5 from E. coli. Individual values (•) and median (―) are given, - -: threshold for positivity. Significant as compared to AIH without therapy: *P  < 0.05; **P  < 0.01; ***P  < 0.001; ****P  < 0.0001. Graphical Abstract [ABSTRACT FROM AUTHOR]

T cells are main actors of the immune system with an essential role in protection against pathogens and cancer. The molecular key event involved in this absolutely central task is the interaction of membrane-bound specific T cell receptors with peptide-MHC complexes which initiates T cell priming, activation and recall, and thus controls a range of downstream functions. While textbooks teach us that the repertoire of mature T cells is highly diverse, it is clear that this diversity cannot possibly cover all potential foreign peptides that might be encountered during life. TCR cross-reactivity, i.e. the ability of a single TCR to recognise different peptides, offers the best solution to this biological challenge. Reports have shown that indeed, TCR cross-reactivity is surprisingly high. Hence, the T cell dilemma is the following: be as specific as possible to target foreign danger and spare self, while being able to react to a large spectrum of bodythreatening situations. This has major consequences for both autoimmune diseases and cancer, and significant implications for the development of T cell-based therapies. In this review, we will present essential experimental evidence of T cell cross-reactivity, implications for two opposite immune conditions, i.e. autoimmunity vs cancer, and how this can be differently exploited for immunotherapy approaches. Finally, we will discuss the tools available for predicting cross-reactivity and how improvements in this field might boost translational approaches. [ABSTRACT FROM AUTHOR]

T cells are main actors of the immune system with an essential role in protection against pathogens and cancer. The molecular key event involved in this absolutely central task is the interaction of membrane-bound specific T cell receptors with peptide-MHC complexes which initiates T cell priming, activation and recall, and thus controls a range of downstream functions. While textbooks teach us that the repertoire of mature T cells is highly diverse, it is clear that this diversity cannot possibly cover all potential foreign peptides that might be encountered during life. TCR cross-reactivity, i.e. the ability of a single TCR to recognise different peptides, offers the best solution to this biological challenge. Reports have shown that indeed, TCR cross-reactivity is surprisingly high. Hence, the T cell dilemma is the following: be as specific as possible to target foreign danger and spare self, while being able to react to a large spectrum of bodythreatening situations. This has major consequences for both autoimmune diseases and cancer, and significant implications for the development of T cell-based therapies. In this review, we will present essential experimental evidence of T cell cross-reactivity, implications for two opposite immune conditions, i.e. autoimmunity vs cancer, and how this can be differently exploited for immunotherapy approaches. Finally, we will discuss the tools available for predicting cross-reactivity and how improvements in this field might boost translational approaches. [ABSTRACT FROM AUTHOR]

Key Points: Question: Is treatment with CD19-targeting–chimeric antigen receptor (CAR) T cells feasible and effective in antisynthetase syndrome, a rare autoimmune disorder characterized by myositis and interstitial lung disease? Findings: CD19-targeting CAR T-cell therapy followed by mycophenolate mofetil was safe, and it induced clinical and serological remission in a patient with progressive antisynthetase syndrome whose condition was refractory to available treatments (including rituximab and azathioprine). Meaning: B-cell depletion and cotargeting of plasmablasts with CD19-targeting CAR T cells deeply resets B-cell immunity, providing a potentially useful tool to treat antisynthetase syndrome. Importance: Autoimmune disorders can affect various organs and if refractory, can be life threatening. Recently, CD19-targeting–chimeric antigen receptor (CAR) T cells were efficacious as an immune suppressive agent in 6 patients with refractory systemic lupus erythematosus and in 1 patient with antisynthetase syndrome. Objective: To test the safety and efficacy of CD19-targeting CAR T cells in a patient with severe antisynthetase syndrome, a complex autoimmune disorder with evidence for B- and T-cell involvement. Design, Setting, and Participants: This case report describes a patient with antisynthetase syndrome with progressive myositis and interstitial lung disease refractory to available therapies (including rituximab and azathioprine), who was treated with CD19-targeting CAR T cells in June 2022 at University Hospital Tübingen in Tübingen, Germany, with the last follow-up in February 2023. Mycophenolate mofetil was added to the treatment to cotarget CD8+ T cells, hypothesized to contribute to disease activity. Exposure: Prior to treatment with CD19-targeting CAR T cells, the patient received conditioning therapy with fludarabine (25 mg/m2 [5 days before until 3 days before]) and cyclophosphamide (1000 mg/m2 [3 days before]) followed by infusion of CAR T cells (1.23×106/kg [manufactured by transduction of autologous T cells with a CD19 lentiviral vector and amplification in the CliniMACS Prodigy system]) and mycophenolate mofetil (2 g/d) 35 days after CD19-targeting CAR T-cell infusion. Main Outcomes and Measures: The patient's response to therapy was followed by magnetic resonance imaging of the thigh muscle, Physician Global Assessment, functional muscle and pulmonary tests, and peripheral blood quantification of anti-Jo-1 antibody levels, lymphocyte subsets, immunoglobulins, and serological muscle enzymes. Results: Rapid clinical improvement was observed after CD19-targeting CAR T-cell infusion. Eight months after treatment, the patient's scores on the Physician Global Assessment and muscle and pulmonary function tests improved, and there were no detectable signs of myositis on magnetic resonance imaging. Serological muscle enzymes (alanine aminotransferase, aspartate aminotransferase, creatinine kinase, and lactate dehydrogenase), CD8+ T-cell subsets, and inflammatory cytokine secretion in the peripheral blood mononuclear cells (interferon gamma, interleukin 1 [IL-1], IL-6, and IL-13) were all normalized. Further, there was a reduction in anti-Jo-1 antibody levels and a partial recovery of IgA (to 67% of normal value), IgG (to 87%), and IgM (to 58%). Conclusions and Relevance: CD19-targeting CAR T cells directed against B cells and plasmablasts deeply reset B-cell immunity. Together with mycophenolate mofetil, CD19-targeting CAR T cells may break pathologic B-cell, as well as T-cell responses, inducing remission in refractory antisynthetase syndrome. This case report summarizes treatment of a patient with antisynthetase syndrome using CD19-targeting T cells against B cells and plasmablasts. [ABSTRACT FROM AUTHOR]

Objectives To evaluate the effect of autologous stem cell transplantation (aSCT) on functional antibodies (abs) to the angiotensin II type-1-receptor (AT1R) and topoisomerase-I (topo-I) in SSc-patients and to analyse their prognostic relevance. Material and methods Forty-three SSc-patients in whom aSCT was performed were analysed. Thirty-one patients had a favourable outcome after aSCT (group 1), 12 patients showed no response or relapse (group 2). Patients' sera were tested for anti-AT1R and anti-topo-I antibodies by ELISA and in a luminometric assay (LA) using AT1R-expressing Huh7-cells for inhibitory or stimulatory anti-AT1R antibodies before and after aSCT (4–217 months, median 28 months). Anti-topo-I antibodies were also analysed for their capacity to inhibit enzyme function. Results A total of 70% of the SSc patients had anti-topo-I- and 51% anti-AT1R antibodies in the ELISA before aSCT. In all instances, anti-topo-I antibodies inhibited topo-I-enzyme function. In the LA, 40% had stimulatory and 12% inhibitory anti-AT1R antibodies. Anti-topo-I- and anti-AT1R-reactivity (ELISA) significantly decreased after aSCT. Before aSCT, anti-topo-I-reactivity was significantly higher in group 2 patients than in group 1 patients (P  < 0.001), while there was no difference between both groups for anti-AT1R antibodies detected by ELISA. Stimulatory anti-AT1R antibodies detected by LA were confined to group 1-patients. Conclusions Reactivity of functionally active anti-AT1R antibodies was not influenced by aSCT, while anti-topo-I antibodies decreased after aSCT. The fact that anti-topo-I antibodies inhibited enzyme function in all instances supports the hypothesis of a pathogenetic role of the topo-I antigen/antibody-system in SSc. High anti-topo-I reactivity before aSCT was associated with an unfavourable, presence of stimulatory anti-AT1R antibodies with a favourable course after aSCT. [ABSTRACT FROM AUTHOR]

The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease lacking specific therapies. This study reports on the identification, characterization, and immunotherapeutic application of HLA-presented neoantigens specific for the DNAJB1-PRKACA fusion transcript in fibrolamellar hepatocellular carcinoma. DNAJB1-PRKACA-derived HLA class I and HLA class II ligands induce multifunctional cytotoxic CD8+ and T-helper 1 CD4+ T cells, and their cellular processing and presentation in DNAJB1-PRKACA expressing tumor cells is demonstrated by mass spectrometry-based immunopeptidome analysis. Single-cell RNA sequencing further identifies multiple T cell receptors from DNAJB1-PRKACA-specific T cells. Vaccination of a fibrolamellar hepatocellular carcinoma patient, suffering from recurrent short interval disease relapses, with DNAJB1-PRKACA-derived peptides under continued Poly (ADP-ribose) polymerase inhibitor therapy induces multifunctional CD4+ T cells, with an activated T-helper 1 phenotype and high T cell receptor clonality. Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion. The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease with limited therapeutic options. Here, the authors identify the DNAJB1-PRKACA protein as a source for immunogenic neoepitopes and a potential target of T cell-based immunotherapy. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade (ICB) is standard-of-care for patients with metastatic melanoma. It may re-invigorate T cells recognizing tumors, and several tumor antigens have been identified as potential targets. However, little is known about the dynamics of tumor antigen-specific T cells in the circulation, which might provide valuable information on ICB responses in a minimally invasive manner. Here, we investigated individual signatures composed of up to 167 different melanoma-associated epitope (MAE)-specific CD8+ T cells in the blood of stage IV melanoma patients before and during anti-PD-1 treatment, using a peptide-loaded multimer-based high-throughput approach. Additionally, checkpoint receptor expression patterns on T cell subsets and frequencies of myeloid-derived suppressor cells and regulatory T cells were quantified by flow cytometry. Regression analysis using the MAE-specific CD8+ T cell populations was applied to identify those that correlated with overall survival (OS). The abundance of MAE-specific CD8+ T cell populations, as well as their dynamics under therapy, varied between patients. Those with a dominant increase of these T cell populations during PD-1 ICB had a longer OS and progression-free survival than those with decreasing or balanced signatures. Patients with a dominantly increased MAE-specific CD8+ T cell signature also exhibited an increase in TIM-3+ and LAG-3+ T cells. From these results, we created a model predicting improved/reduced OS by combining data on dynamics of the three mostinformative MAE-specific CD8+ T cell populations. Our results provide insights into the dynamics of circulating MAE-specific CD8+ T cell populations during ICB, and should contribute to a better understanding of biomarkers of response and anticancer mechanisms. [ABSTRACT FROM AUTHOR]

Objectives: 1) To detect functionally active antibodies(abs) to the angiotensin II type-1-receptor (AT1R) by a novel luminometric assay. 2) To assess their prevalence in systemic sclerosis (SSc), other collagen disorders, as well as in further chronic inflammatory disorders including autoimmune, toxic and chronic viral diseases. 3) To compare these abs with anti-AT1R antibodies by ELISA as well as with antibodies to endothelin-type-A receptors (ETA1) and to topoisomerase I (topo-I) with respect to their specificity and clinical relevance. Methods: Sera from 98 SSc-patients, 110 patients with other chronic inflammatory rheumatic disorders, 97 patients with autoimmune liver diseases, 57 patients with toxic or chronic viral liver diseases and 36 healthy controls were analyzed. A luminometric bioassay was established with Huh-7-cells constitutively expressing the AT1R. Patients' sera were also tested by commercially available ELISA for anti-AT1R, -ETA1- and by an in-house ELISA for anti–topo-I-abs. Results: Fifty-two percent of the SSc-patients had functionally active anti-AT1R-abs with stimulatory (34%) or inhibitory capacity (18%). They were present also in up to 59% of patients with other rheumatic diseases but only 22% of healthy individuals (sensitivity 52%, specificity 53%). The functionally active antibodies detected by the luminometric assay did not correlate with anti-AT1R-, -ETA1- or -topo-I-abs measured by ELISA, but there was a strong correlation between anti-topo-I-, AT1R-, and -ETA1-ab reactivity measured by ELISA. Sensitivities of 55%, 28% and 47% and specificities of 66%, 87%, and 99% were calculated for these anti-AT1R-, -ETA1-, and anti-topo-I-abs, respectively. Functionally active abs did not correlate with disease severity or any organ manifestation. In contrast, abs to topo-I, AT1R, and ETA1 were associated with digital ulcers, pulmonary- and esophageal manifestation. Conclusions: Functionally active anti-AT1R-abs can be detected in SSc-patients but do not correlate with disease activity. They are not specific for this disease and occur also in other autoimmune disorders and even viral or toxic diseases. Also, the vascular antibodies detected by ELISA are not SSc-specific but correlated with disease manifestations. In contrast, anti-topo-I-abs were confirmed to be a highly specific biomarker for both, diagnosis and organ manifestations of SSc. [ABSTRACT FROM AUTHOR]

Low human leukocyte antigen (HLA)-DR expression might compromise CD4+ T-cell-mediated anti-tumor immunity. Its immunological and clinical significance however remain undefined in non-promyelocytic acute myeloid leukemia (AML). Taking advantage of mass spectrometry-based immunopeptidome analysis of primary AML samples (n = 31), we studied the implications of low HLA-DR expression for antigen presentation and analyzed its association with disease characteristics and survival within a cohort of 399 AML patients. Remarkably, overall HLA-DR/DQ immunopeptidome diversity was preserved in AML with low HLA-DR expression (HLA-DRlow AML) and was associated with a shift in HLA-DR/DQ allotype abundances (HLA-DQ to HLA-DR/DQ ligand ratio 0.36 vs 0.19 in HLA-DRlow and HLA-DRhigh patients, respectively). Consistent with unimpaired antigenicity, survival was similar in HLA-DRlow and HLA-DRhigh patients. Demonstrating for the first time that overall HLA-DR/DQ antigen presentation is preserved in HLA-DRlow AML, our findings provide a rationale for the non-inferior outcome observed in HLA-DRlow AML patients. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a severe autoimmune disease of unknown etiology. The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are stress-inducible cell surface molecules. MICA and MICB label malfunctioning cells for their recognition by cytotoxic lymphocytes such as natural killer (NK) cells. Alterations in this recognition have been found in SLE. MICA/MICB can be shed from the cell surface, subsequently acting either as a soluble decoy receptor (sMICA/sMICB) or in CD4+ T-cell expansion. Conversely, NK cells are frequently defective in SLE and lower NK cell numbers have been reported in patients with active SLE. However, these cells are also thought to exert regulatory functions and to prevent autoimmunity. We therefore investigated whether, and how, plasma membrane and soluble MICA/B are modulated in SLE and whether they influence NK cell activity, in order to better understand how MICA/B may participate in disease development. We report significantly elevated concentrations of circulating sMICA/B in SLE patients compared with healthy individuals or a control patient group. In SLE patients, sMICA concentrations were significantly higher in patients positive for anti-SSB and anti-RNP autoantibodies. In order to study the mechanism and the potential source of sMICA, we analyzed circulating sMICA concentration in Behcet patients before and after interferon (IFN)-α therapy: no modulation was observed, suggesting that IFN-α is not intrinsically crucial for sMICA release in vivo. We also show that monocytes and neutrophils stimulated in vitro with cytokines or extracellular chromatin up-regulate plasma membrane MICA expression, without releasing sMICA. Importantly, in peripheral blood mononuclear cells from healthy individuals stimulated in vitro by cell-free chromatin, NK cells up-regulate CD69 and CD107 in a monocyte-dependent manner and at least partly via MICA-NKG2D interaction, whereas NK cells were exhausted in SLE patients. In conclusion, sMICA concentrations are elevated in SLE patients, whereas plasma membrane MICA is up-regulated in response to some lupus stimuli and triggers NK cell activation. Those results suggest the requirement for a tight control in vivo and highlight the complex role of the MICA/sMICA system in SLE. [ABSTRACT FROM AUTHOR]

The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, we treated 2 patients with defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved during treatment. Moreover, coagulation parameters indicating hypofibrinolysis and complement activation normalized. The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, 2 patients received defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved and hypofibrinolysis/complement activation normalized during treatment. [ABSTRACT FROM AUTHOR]

Background and Aims: In primary biliary cholangitis (PBC), antibodies to a peptide of the muscarinic acetylcholine receptor 3 (mAChR3) have been described. Since the mAChR3 is expressed on cholangiocytes and mAChR3-signaling is involved in the pathogenesis of chronic inflammatory biliary diseases, we wanted to investigate whether anti-mAChR3-antibodies influence the function of the receptor and the proliferative response of cholangiocytes. Methods: Immunoglobulins were isolated by ammonium sulfate precipitation using sera from patients with PBC (n = 63) and with other chronic liver disorders (n = 150). All immunoglobulins were analyzed by a luminometric assay using Chinese hamster ovary (CHO) cells overexpressing the mAChR3 and cholangiocytes (TFK-1-cells) expressing the receptor constitutively. Cell proliferation was measured by 3H-thymidine assay. PBC patients were also analyzed in the follow-up. Results: Antibodies inhibiting the mAChR3 were found in 49 and 79% of PBC patients using CHO-cells or TFK-1-cells, respectively, but only in up to 26% of controls (p < 0.01). Stimulatory antibodies were hardly detected. Antibody reactivity only marginally changed during the course of the disease, independently of the choice of treatment (ursodeoxycholic acid, immunosuppressive therapy, or no medication). There was no correlation with laboratory, clinical or histological parameters, but the antibodies were more frequently found in PBC patients with a benign course (96%) than in patients with active disease progressing to late stages within 10 years (57%; p < 0.01). Proliferation of cells was not influenced by immunoglobulins from PBC-patients. Conclusion: Sera from patients with PBC contain inhibitory antibodies to the mAChR3 on cholangiocytes (TFK-1 cells) without influencing TFK-1-cell proliferation. These antibodies were predominantly observed in patients with non-progressing PBC. [ABSTRACT FROM AUTHOR]

Background: Determination and quantification of serum free light chains (FLC) is essential for the diagnosis and monitoring of patients with monoclonal gammopathies. Currently, the Freelite assay (The Binding Site, United Kingdom) and the N Latex FLC assay (Siemens Healthineers, Germany) are available for FLC determination. Data concerning stability of FLC following long-term storage are limited. Therefore, the aim of the present study is to investigate the stability of FLC in frozen samples determined by the N latex FLC assay. Methods: One hundred eighty-eight serum samples from 47 patients with monoclonal gammopathies were analyzed at the beginning and after long-term storage (-20°C, 193 - 568 days). Serum free light chain kappa (κFLC) and lambda (λFLC) concentrations were measured, and the corresponding kappa/lambda (κ/λ FLC) ratios were calculated. All samples were analyzed with the N latex FLC assay on a single BNII System. Results: Comparison analyses between fresh and stored samples revealed very strong correlations for the determination of κFLC (r = 0.993), λFLC (r = 0.998) and the calculated κ/λ FLC ratio (r = 0.997). Median percentage changes of κFLC (-0.5%) and λFLC (-0.8%) measurements and the calculated κ/λ FLC ratios (-3.7%) were within the calculated analytical imprecision of the FLC assay. Changes of κFLC (r = 0.17, p = 0.02) and λFLC (r = 0.28, p < 0.01) concentrations and of the κ/λ FLC ratio (r = 0.18, p = 0.01) over time were very weak, but statistically significant. Conclusions: Serum free light chains in serum samples are sufficiently stable following long-term frozen storage and are therefore suitable to be measured with the N Latex FLC assay. [ABSTRACT FROM AUTHOR]

Recent studies suggest that CLL is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell-based immunotherapy. However, CLL is associated with a profound immune defect, which might represent a critical limitation for mounting clinically effective antitumor immune responses. As several studies have demonstrated that lenalidomide can reinforce effector T-cell responses in CLL, the combination of T-cell-based immunotherapy with the immunomodulatory drug lenalidomide represents a promising approach to overcome the immunosuppressive state in CLL. Antigen-specific immunotherapy also requires the robust presentation of tumor-associated HLA-presented antigens on target cells. We thus performed a longitudinal study of the effect of lenalidomide on the HLA ligandome of primary CLL cells in vitro. We showed that lenalidomide exposure does not affect absolute HLA class I and II surface expression levels on primary CLL cells. Importantly, semi-quantitative mass spectrometric analyses of the HLA peptidome of three CLL patient samples found only minor qualitative and quantitative effects of lenalidomide on HLA class I- and II-restricted peptide presentation. Furthermore, we confirmed stable presentation of previously described CLL-associated antigens under lenalidomide treatment. Strikingly, among the few HLA ligands showing significant modulation under lenalidomide treatment, we identified upregulated IKZF-derived peptides, which may represent a direct reflection of the cereblon-mediated effect of lenalidomide on CLL cells. Since we could not observe any relevant influence of lenalidomide on the established CLL-associated antigen targets of anticancer T-cell responses, this study validates the suitability of lenalidomide for the combination with antigen-specific T-cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

Objectives. To evaluate the effect of autologous stem cell transplantation (aSCTrans) on antibody (Ab) reactivity towards topo I in patients with SSc, and to see whether it may correlate with clinical outcome after aSCTrans. Methods. Eighteen anti-topo/Scl70-positive patients with SSc in whom non-myeloablative aSCTrans had been performed were analysed. Seven patients showed good response without relapse for several years (group 1), eight primarily responded but later relapsed and three did not respond (group 2). A total of 74 sera were analysed at different time points and tested by ELISA against full length (fl) topo I, truncated (tr) topo I and a previously identified immunodominant epitope covering amino acid 489-573. Results. Eighty-three percent had IgG Abs to topofl and topotr. Ab reactivity significantly decreased after aSCTrans, but remained positive in 10 of the 11 patients followed for up to 24 months. The decrease did not correlate with the clinical outcome after aSCTrans. Fifty-six percent of the patients reacted with topo489-573, and reactivity was nearly confined to group 2. There was no correlation between Ab reactivity towards topofl or topo489-573 and the modified Rodnan Skin Score before aSCTrans or its decrease after aSCTrans. Conclusions. Although aSCTrans is a good treatment option in patients with progressive SSc, it does not abrogate Ab reactivity towards topo I. The presence of anti-topo489-573 Abs before aSCTrans may indicate a less favourable course after aSCTrans. [ABSTRACT FROM AUTHOR]

This report discusses the history and mechanisms of vaccination of humans as well as the engineering of therapeutic antibodies. Deeper understanding of the molecular interactions involved in both acquired and innate immunity is allowing sophistication in design of modified and even synthetic vaccines. Recombinant DNA technologies are facilitating development of DNA-based vaccines, for example, with the recognition that unmethylated CpG sequences in plasmid DNA will target Toll-like receptors on antigen-presenting cells. Formulations of DNA vaccines with increased immunogenicity include engineering into plasmids with 'genetic adjuvant' capability, incorporation into polymeric or magnetic nanoparticles, and formulation with cationic polymers and other polymeric and non-polymeric coatings. Newer methods of delivery, such as particle bombardment, DNA tattooing, electroporation, and magnetic delivery, are also improving the effectiveness of DNA vaccines. RNA-based vaccines and reverse vaccinology based on gene sequencing and bioinformatic approaches are also considered. Structural vaccinology is an approach in which the detailed molecular structure of viral epitopes is used to design synthetic antigenic peptides. Virus-like particles are being designed for vaccine deliveries that are based on structures of viral capsid proteins and other synthetic lipopeptide building blocks. A new generation of adjuvants is being developed to further enhance immunogenicity, based on squalene and other oil-water emulsions, saponins, muramyl dipeptide, immunostimulatory oligonucleotides, Toll-like receptor ligands, and lymphotoxins. Finally, current trends in engineering of therapeutic antibodies including improvements of antigen-binding properties, pharmacokinetic and pharmaceutical properties, and reduction of immunogenicity are discussed. Taken together, understanding the chemistry of vaccine design, delivery and immunostimulation, and knowledge of the techniques of antibody design are allowing targeted development for the treatment of chronic disorders characterized by continuing activation of the immune system, such as autoimmune disorders, cancer, or allergies that have long been refractory to conventional approaches. [ABSTRACT FROM AUTHOR]

This work compiles information on the principles of diagnostic immunochemical methods and the recent advances in this field. It presents an overview of modern techniques for the production of diagnostic antibodies, their modification with the aim of improving their diagnostic potency, the different types of immunochemical detection systems, and the increasing diagnostic applications for human health that include specific disease markers, individualized diagnosis of cancer subtypes, therapeutic and addictive drugs, food residues, and environmental contaminants. A special focus lies in novel developments of immunosensor techniques, promising approaches to miniaturized detection units and the associated microfluidic systems. The trends towards high-throughput systems, multiplexed analysis, and miniaturization of the diagnostic tools are discussed. It is also made evident that progress in the last few years has largely relied on novel chemical approaches. [ABSTRACT FROM AUTHOR]

Background: Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. Methods: Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. Results: Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. Conclusion: This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug clearance in patients on Tmab therapy. [ABSTRACT FROM AUTHOR]

Background: Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) constitute rare chronic inflammatory biliary diseases which likely comprise genetic, environmental and autoimmune factors. Specific inhibitory (auto-) antibodies against the muscarinic acetylcholine receptor type 3 (mAChR3 auto-ab) may contribute to the pathogenesis of chronic biliary inflammation by modulating mAChR3− mediated signaling. Aims: The aim of this study was to analyze the prevalence and relevance of inhibitory mAChR3 auto-ab (mAChR3inh+ auto-ab) in a large cohort of PBC patients from two independent tertiary centers in Berlin and Leipzig in comparison to a large PSC cohort. Baseline parameters and response rates to standard treatment with ursodeoxycholic acid (UDCA) were characterized with respect to the individual mAChR3 auto-ab status. Methods: In total, the study population comprised 437 PBC patients, 187 PSC patients and 80 healthy controls. Clinical and laboratory baseline characteristics were retrieved from medical records. The response to ursodeoxycholic acid (UDCA) therapy after 12 months of treatment was available in 176 PBC and 45 PSC patients. Results: The prevalence of mAChR3inh+ auto-ab was significantly higher among PBC patients (11.2%, 49/437; p = 0.008 vs. healthy controls) and PSC patients (33.6%, 63/187; p < 0.0001 vs. healthy controls) compared to healthy controls (2.5%, 2/80), respectively. PBC patients with mAChR3inh+ auto-ab exhibited significantly higher levels of alkaline phosphatase (ALP) and bilirubin, which constitute established parameters for PBC risk stratification. Moreover, mAChR3inh+ PBC patients tended to show decreased response rates to UDCA therapy compared to PBC patients without mAChR3inh+ auto-ab (mAChR3− PBC). In contrast, PSC patients with mAChR3inh+ auto-ab showed no significant differences in laboratory findings compared to mAChR3 auto-ab negative (mAChR3−) PSC patients. Conclusion: MAChR3inh+ auto-ab might be involved in the pathogenesis and treatment response of chronic biliary inflammation in patients with PBC but not in patients with PSC. [ABSTRACT FROM AUTHOR]

CD4+ T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide-based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors. Here we describe the first immunological evaluation of a survivin-derived CD4+ T cell epitope in a multipeptide immunotherapy trial for prostate carcinoma patients. The survivin peptide is promiscuously presented by several human HLA-DRB1 molecules and, most importantly, is naturally processed by dendritic cells. In vaccinated patients, it was able to induce frequent, robust and multifunctional CD4+ T cell responses, as monitored by IFN-γ ELISPOT and intracellular cytokine staining. Thus, this HLA-DR restricted epitope is broadly immunogenic and should be valuable for stimulating T helper cells in patients suffering from a wide range of tumors. [ABSTRACT FROM AUTHOR]

The primary objective of this "Glossary of Terms Used in Immunotoxicology" is to give clear definitions for those who contribute to studies relevant to immunotoxicology but are not themselves immunologists. This applies especially to chemists who need to understand the literature of immunology without recourse to a multiplicity of other glossaries or dictionaries. The glossary includes terms related to basic and clinical immunology insofar as they are necessary for a self-contained document, and particularly terms related to diagnosing, measuring, and understanding effects of substances on the immune system. The glossary consists of about 1200 terms as primary alphabetical entries, and Annexes of common abbreviations, examples of chemicals with known effects on the immune system, autoantibodies in autoimmune disease, and therapeutic agents used in autoimmune disease and cancer. The authors hope that among the groups who will find this glossary helpful, in addition to chemists, are toxicologists, pharmacologists, medical practitioners, risk assessors, and regulatory authorities. In particular, it should facilitate the worldwide use of chemistry in relation to occupational and environmental risk assessment. [ABSTRACT FROM AUTHOR]

Background: A high incidence of autoantibodies to lens proteins has been found in sera of patients with uveitis. We showed previously that the anti-lens antibodies reacted predominantly with α-crystallins. The aim of the present study was to identify immunodominant epitopes within the protein chains of human αA- and αB-crystallin. Methods: Epitope specificities of antibodies to αA- and αB-crystallin were examined by ELISA using synthetic overlapping peptides, spanning the entire length of both α-crystallins. The peptides consisted of 25 amino acid residues, with an overlap of at least eight amino acids each. The synthetic peptides were tested against sera of 110 patients with different uveitis forms, classified according to anatomical location of intraocular inflammation. Results: Four immunodominant regions within the protein chains of αA- and αB-crystallin could be identified. These regions were recognized by antibodies in sera of 56% of uveitis patients. Anti-lens antibodies of IgG-type reacted preferentially with regions located at amino acid (aa) residues aa:69-93 and aa:137-161 of αA-crystallin as well as aa:69-110 and aa:137-161 of αB-crystallin. IgM antibodies recognized predominantly region aa:149-173 of αA-crystallin, and aa:69-110 and aa:151-175 of αB-crystallin. IgM antibodies directed to peptide aa:69-93 of αB-crystallin were found in sera of 30% of patients with intermediate uveitis. Conclusions: Four immunodominant B-cell epitopes within the protein chains of αA- and αB-crystallin have been identified; however, no clear correlation with the anatomically defined uveitis subtypes has been found except for intermediate uveitis. Whether there may be a correlation with uveitis forms with similar etiopathogenesis has to be evaluated in further studies. [ABSTRACT FROM AUTHOR]

Background and Aims Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody ( AMA) negative sera from patients with primary biliary cirrhosis ( PBC) has been shown to contain parts of the five F1- ATPase subunits α, β, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC. Methods Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis ( AIH), 35 patients with primary sclerosing cholangitis ( PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases ( IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F1- ATPase-subunits α-δ were recombinantly expressed in E scherichia coli, purified and applied to ELISA and Western blotting. Results In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F1-subunits α-δ. The β- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-β- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-β and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the β- and only 6% to the γ-subunit. Sera from healthy blood donors were negative. Conclusions Antibodies to the β- and γ-subunits of F1- ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver. [ABSTRACT FROM AUTHOR]

Background: In a recent study we showed that in patients with primary biliary cirrhosis (PBC) being positive or negative for anti-M2 antibodies reacting with the 2-oxoacid-dehydrogenase complex (ODC) also antibodies to the beta- and gamma-subunits of F1F0-ATPase (anti-β, anti-γ) occur. This is a mitochondrial enzyme but parts are also expressed on plasma membranes of endothelial cells. Here we wanted to analyse in more detail their clinical relevance. Methods: Fifty-nine untreated and histologically defined PBC patients who had been followed for at least five years were included into the study (51 anti-M2 positive, 8 anti-M2 negative). Twenty-three of them were treated in the follow up with ursodeoxycholic acid (UDCA), eight received during a trial methotrexate (MTX). In 13 patients orthotopic liver transplantation (OLT) had to be performed. Serum samples before and during therapy were available. Patients were analysed with respect to laboratory parameters, disease activity and histological stages. Patients' sera were tested by ELISA for IgG- and IgM-antibodies against the beta- and gamma-subunits which had been recombinant expressed in E.coli and highly purified by electro-elution from SDS-gels after electrophoresis. Results: Fifty-nine percent of the anti-M2 positive and 50% of the anti-M2 negative PBC patients had anti-β- and/or anti-γ-antibodies. There were no differences between anti-β- and/or anti-γ-antibody positive or negative patients with respect to biochemical parameters, immunoglobulins, histological stages or disease activity. Antibody reactivity significantly decreased during UDCA and MTX-treatment and also after OLT. Conclusions: Antibodies to the β- and γ-subunits of F1F0-ATPase occur in anti-M2 positive and -negative PBC but do not have any relevance with respect to clinical activity or prognosis. However, in contrast to the anti-M2 antibodies they decrease during UDCA and immunosuppressive therapy. [ABSTRACT FROM AUTHOR]

Knowledge of natural mistletoe lectins (nML) pharmacokinetics can be regarded as essential for further rational studies with mistletoe preparations. Studies with intravenous application of a recombinant type II ribosome inactivating protein (rML) analogous to nML revealed a short half-life of about 13 min in cancer patients. This open-label, phase I, monocenter clinical trial was performed in order to describe the pharmacokinetics of nML. In 15 healthy male volunteers aged 18–42 years, nML were detected with a modified sandwich immuno-polymerase chain reaction (PCR) technique (Imperacer®, Chimera Biotec) after single subcutaneous injection of a mistletoe extract (abnobaVISCUM® Fraxini 20 mg) with marketing authorization containing about 20 µg nML/ml. Secondary objectives were safety and the number of activated natural killer cells (CD54+/CD94+). In none of the volunteers were nML detectable before the injection, and in all volunteers, nML were detected in serum samples after injection. Individual variability, however, was large. Mean and median peak concentrations were reached 1 and 2 h after injection, respectively. In some volunteers, nML were still detectable at the final investigation 2 weeks after injection. The injection resulted in fever and flu-like symptoms in all volunteers, but no serious adverse events occurred. All symptoms and local reactions at the injection site completely disappeared within a range of 4–95 days. The number of activated natural killer (NK) cells did not change. Natural ML from abnobaVISCUM® Fraxini 20 mg are detectable in serum after a single subcutaneous injection. Detectability is considerably longer compared with intravenously administered rML. The subcutaneous injection of this preparation without usual pretreatment with lower doses results in short-lasting fever and other flu-like symptoms. [ABSTRACT FROM AUTHOR]

Phenprocoumon-induced liver injury is a rare complication of oral anticoagulation. The mechanisms leading to this side effect are not entirely clear. Here we present data that at least in a subgroup of patients in whom phenprocoumon-induced liver disease was suspected, immunological processes may play an important role. Thirty patients with suspected phenprocoumon-induced liver disease from different hospitals in Germany were analyzed. Peripheral blood mononuclear cells (PBMC) of these patients were tested in the lymphocyte transformation test (LTT) for reactivity with phenprocoumon in vitro. As controls, PBMC were isolated from ten individuals treated with phenprocoumon but without any side effects, and from ten healthy individuals who have never received the drug. Fifteen of the 30 patients had sensitized lymphocytes toward phenprocoumon as shown by LTT. Four patients had taken the drug for more than 5 years; in one patient, liver disease appeared after 1 day of phenprocoumon intake. There was no correlation between a positive LTT and clinical/laboratory parameters. None of the 20 controls had sensitized lymphocytes toward phenprocoumon. Applying the LTT, we were able to unravel the cause of suspected phenprocoumon-induced liver injury as a drug allergic reaction in 15 out of 30 analyzed patients. [ABSTRACT FROM AUTHOR]

Background: In primary biliary cirrhosis (PBC), autoreactivity mainly targets members of the pyruvate dehydrogenase complex (PDC). Because PDC subunits are expressed on the surface of mycoplasma and molecular mimicry may be one aetiological factor, we analysed the presence of mammalian and mycoplasma PDC-specific antibodies in PBC patients. Methods: Antibodies to porcine PDC and Mycoplasma pneumoniae (mp) antigens mpPDH-C (to be designated mpPDC-E2 chain), mpPDH-B (to be designated mpPDC-E1β chain), mpCARDS TX and mpP1 were investigated in sera from 43 PBC patients, 19 patients with autoimmune hepatitis and 11 healthy controls by an enzyme-linked immunosorbent assay and Western blotting. To study the rate of acute mycoplasma infection, an adhesin P1-specific polymerase chain reaction (PCR) was performed. Results: Immune reactivity to the mpPDC-E2 antigen was significantly enhanced in PBC patients (83.7%) as compared with controls (overall frequency of 36.7%), while antibodies to the porcine PDC-E2 chain were found only in PBC patients (88%) excluding a simple cross-reactivity of PDC-related antibodies. This observation was confirmed by inhibition studies demonstrating that porcine PDC did not inhibit mycoplasma PDC-specific antibodies and vice versa. The occurrence of antibodies to mpPDC seems to precede the occurrence of antibodies to porcine PDC. Infection with mycoplasma was equally distributed in the groups as evidenced by an antibody frequency comparable to CARDS TX and P1 and PCR reactivity. Conclusion: Because PBC patients show a significantly enhanced frequency of mpPDC-E2-related antibodies, besides other factors, molecular mimicry between surface molecules of mycoplasma and epitopes of the autoantigen may play a central role in the aetiopathology of PBC. [ABSTRACT FROM AUTHOR]

The success of allogeneic stem cell transplantation depends upon the degree of human leukocyte antigen (HLA) compatibility between the donor and patient since T-cell responses to foreign HLA molecules can result in aggressive immune responses. The human HLA locus on chromosome 6 is highly polymorphic within the population. Due to the large number of allels, the exact determination of the individual HLA polymorphisms (HLA typing) is a technically challenging task requiring different molecular biological procedures. [ABSTRACT FROM AUTHOR]

The objectives of the study were to evaluate the prevalence of antinuclear antibodies (ANA) in patients with fibromyalgia (FM) and the probability of the development of clinically overt connective tissue diseases. Four hundred and fifty FM patients were compared to 129 healthy matched blood donors. ANA testing was performed by immunofluorescence on rat tissue sections; in case of highly positive results, ANA were specified further by ELISA and immunodiffusion. All ANA positive FM patients were invited for a control examination. The ANA negative patients received a questionnaire, which was designed to identify those patients with possible connective tissue diseases (CTD). There was no significant difference in the frequency of ANA or thyroid antibodies between patients and controls (11.6% vs. 7%). Two patients had developed SLE: one was already ANA/anti-dsDNA positive at time of first diagnosis of FM; in the other, specific antibodies and SLE-related symptoms developed after 4.5 years. The probability for FM patients to develop CTD (SLE) within one year is 0.0027%, which is comparable to the incidence of SLE in the general population (0.005%). The risk of CTD is not increased in FM. The detection of ANA does not predict the development of CTD. However, in individual cases, FM may be an early sign of an autoimmune disease. [ABSTRACT FROM AUTHOR]

Abstract Background  Uveitis is an intraocular inflammatory disease in which autoimmune reactions have been discussed in playing an important role. Many data in this respect derived from the animal model of “experimental autoimmune uveitis”, where several organ-specific autoantigens have been described such as the retinal S-antigen and the interphotoreceptor retinoid-binding protein. However, their diagnostic and pathogenic role in humans has been controversially discussed. In recent studies, the possible relevance of βB1-crystallin, present in lens and ciliary body, has been outlined. We, therefore, wanted to analyse whether sera from patients with uveitis might contain antibodies to lens proteins Methods  Human lenses from cadaveric eyes were shock frozen, homogenized, and resuspended. The resulting suspension (human lens protein fraction - HLPF) was analyzed for antigenicity by ELISA and Western blotting with patients’ sera. A total of 165 patients with uveitis, 54 patients with scleritis and episcleritis, 56 patients with other eye diseases, and 112 healthy blood donors were studied. Results   Twenty-six (49%) of the 53 patients with anterior uveitis, 17 (32%) of the 53 patients with intermediate uveitis and 7 (22%) of 32 patients with posterior uveitis reacted in the ELISA with the HLPF. Antibodies to lens antigens were detected in one-third of patients with panuveitis and retinal vasculitis. In contrast, only 12% of the healthy blood donors were positive in the ELISA. The number of patients with an autoimmune response to α-crystallins in Western blot predominated in all investigated groups. Conclusions  These data indicate that antibodies to lens proteins occur in a high incidence in sera from patients with uveitis. Forty-nine percent of the patients with anterior uveitis and only 12% of healthy controls were positive in the ELISA. In our groups of patients and controls the autoantibodies reacted in the Western blot predominantly with α-crystallin. Further studies are required to analyze in more detail the clinical and etiopathogenetic relevance of the antilens antibodies in uveitis. [ABSTRACT FROM AUTHOR]

Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. Patients and methods: M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive ( n=5) or negative ( n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). Results: Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2+/E1+ and the CFT-negative sera as PDC-E2+/E1−. The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1α and E1β antibodies at a frequency of 74% and 67%. Conclusions: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target. [ABSTRACT FROM AUTHOR]

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 β2-glycoprotein I (β2-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-β2- 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. [ABSTRACT FROM AUTHOR]

Background and Objectives: Mistletoe preparations, which are widely used among patients with cancer in Germany, have immunomodulating properties in vitro and in vivo. The aim of this evaluation was to determine and compare the effects of a lectin-rich (Iscador Qu [IQ] special, Weleda Company, Schwäbisch, Gmünd, Germany.) and a lectin-poor but viscotoxin-rich (Iscador Pini [IP] Weleda Company) mistletoe preparation on clinical and hematologic parameters in healthy subjects. Design: In a double-blinded study, 48 volunteers were randomized to one of three groups: 16 received IQ or IP in increasing doses or placebo twice per week subcutaneously for 12 weeks. The differential blood count and the acute phase markers haptoglobin and C-reactive protein were examined weekly and the symptoms were scored using standardized questionnaires. Results: IQ resulted in significant eosinophilia (315 ± 109) beginning at week 5 (until week 12) compared to IP (183 ± 120) or placebo (200 ± 179). Furthermore, the acute phase marker haptoglobin was significantly increased in the IQ group during week 4. Dose-dependent local reactions (LRs) at the injection site occurred in all subjects who received mistletoe preparations but were stronger in the IQ-treated subjects than in the IP-treated group. The LRs observed in the IQ-treated group were characterized by stronger itching and longer latency than LRs in the IP-treated group (p < 0.05). Severe side-effects did not occur in any of the probands. Conclusions: IQ but not IP can induce eosinophilia in healthy individuals, and this may be related to its content of mistletoe lectins. In contrast, exposure to the viscotoxin-enriched extract IP did not result in specific changes of hematologic parameters. Furthermore, intensity and time course of local reactions seemed to depend on the concentration of mistletoe lectins in those extracts. [ABSTRACT FROM AUTHOR]

OBJECTIVE:Retrospective studies have reported that subtypes of antimitochondrial antibodies (AMAs) discriminate between a benign and a progressive course in patients with primary biliary cirrhosis (PBC). Four AMA profiles (A–D) were defined: profiles A and B associated with a benign course and C and D with a progressive course. We aimed to confirm whether AMA profiles predict prognosis in a large sample of North American patients with PBC.METHODS:Stored pretreatment sera from patients with PBC from two centers were tested for AMA profiles using standard techniques. Proportions of patients in each profile group reaching the endpoints of liver transplantation or death from liver disease were compared. Kaplan-Meier curves were constructed comparing AMA profiles.RESULTS:All 472 patients studied had AMA positive, biopsy-confirmed PBC. Mean age at diagnosis was 53 yr, 90% were female, mean follow-up was 7.6 yr (range = 0.5–23), and 51% received ursodeoxycholic acid for >6 months. Profile A was not detected; 16.7% had profile B; 51.1%, profile C; and 32.2%, profile D. Duration of follow-up was comparable among the different profile groups. The proportions of patients reaching endpoints of death from liver disease or transplantation did not differ among the AMA profiles. No difference in the Kaplan-Meier curves between the different profile groups was observed (p > 0.05).CONCLUSION:AMA profiles do not predict prognosis in patients with PBC. [Copyright &y& Elsevier]

Background. Reduction of pathological autoantibodies and circulating immune complexes can be useful in the treatment of autoimmune disease. Plasmapheresis has been shown to reduce autoantibody levels in systemic lupus erythematosus (SLE), but its effect on patients' outcome was not better compared with conventional immunosuppression in the past. [ABSTRACT FROM PUBLISHER]

In 1983, a female patient born in 1963 presented with symptoms of ulcerative colitis and typical clinical and histological signs of primary sclerosing cholangitis (PSC). At this time only pANCA were positive while other marker antibodies for autoimmune liver disorders could not be detected. In summer 1987 the clinical picture changed and was replaced by laboratory and histological signs typical of autoimmune hepatitis (AIH). Thus, IgG levels increased considerably and cholestatic enzymes became normal. For the first time, anti-liver-pancreas antibodies (LP), a diagnostic marker for AIH type III could be detected. In the following years several relapses occurred also induced by repeated discontinuation of immunosuppressive therapy. Symptoms of colitis persisted but signs of cholestasis remained absent for the following ten years. In 1997, colitis exacerbated again and colectomy had to be performed together with liver transplantation. Surprisingly, histology of the explanted liver now showed the typical features of PSC stage III/IV while the significant criteria for AIH were now lacking. Thus, progression to cirrhosis was, probably, mainly induced by the biliary destructive and fibrotic process although biochemical and serological data were clearly indicative of an autoimmune, i.e. AIH-related manifestation. [ABSTRACT FROM AUTHOR]

A patient with inoperable adenocarcinoma of the pancreas was treated with intraperitumoral and peritumoral injections of a Viscum album L. extract containing 5700 ng/mL mistletoe lectin, mainly mistletoe lectin 1 (Abnobaviscum Quercus 2) for 5 weeks (1 injection per week). After the third injection (day 22), a marked eosinophilia was observed (1800 per microliter) that rose to 3268 per microliter after the fifth injection (day 42). Furthermore, histology performed on day 28 revealed accumulation of eosinophils in ductal lesions and adjacent stroma in addition to the features of ductal adenocarcinoma. In order to investigate whether eosinophilia correlated with immunological features, we analyzed cytokine production of peripheral blood mononuclear cells (PBMC) from this patient after stimulation with antigens known to "unmask" an individual's predisposition for defined immunoreactions, namely purified protein derivative (PPD) as a stimulator of T-helper (T[sub H]1)1-cells and tetanus toxoid (TT) as an activator of T[sub H]2-cells. PBMC of the patient showed a strong proliferation and production of interleukin (IL)-5 and IL-10 after incubation with TT indicating a type-2 response. Simultaneously, PBMC were induced to proliferate and produce interferon γ (IFN-γ) by incubation with PPD suggesting also a type-1 response. These data would readily explain the eosinophilia because eosinophils are effector cells of type-2 reaction but also require type-1 cytokines. Although the overall clinical course of the patient was rapidly progressive, temporary stabilization of the patient's general condition during mistletoe treatment was observed. It is, however, still an open question whether this transient benefit was due to the induction of eosinophilia by a type-2 response. Conclusion: Before high-dose intratumoral and peritumoral treatment with a Viscum album L. extract containing mistletoe, lectin 1 can be associated with hypereosinophilia and strong production of T[sub H]1 as T[sub H]2 cytokines as well. [ABSTRACT FROM AUTHOR]

We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-CoV-2-derived peptides performed in March and April 2020, respectively. The first set of peptides contained eight peptides predicted to bind to the individual's HLA molecules. The second set consisted of ten peptides predicted to bind promiscuously to several HLA-DR allotypes. The vaccine formulation contained the new TLR 1/2 agonist XS15 and was administered as an emulsion in Montanide as a single subcutaneous injection. Peripheral blood mononuclear cells isolated from blood drawn before and after vaccinations were assessed using Interferon-γ ELISpot assays and intracellular cytokine staining. We detected vaccine-induced CD4 T cell responses against six out of 11 peptides predicted to bind to HLA-DR after 19 days, following vaccination, for one peptide already at day 12. We used these results to support the design of a T-cell-inducing vaccine for application in high-risk patients, with weakened lymphocyte performance. Meanwhile, an according vaccine, incorporating T cell epitopes predominant in convalescents, is undergoing clinical trial testing. [ABSTRACT FROM AUTHOR]