Search

IVIG preparations consisting of pooled IgG are increasingly used for the treatment of autoimmune diseases. IVIG is known to regulate the viability of immune cells, including neutrophils. We report that plasma-derived IgA efficiently triggers death of neutrophils primed by cytokines or TLR agonists. IgA-mediated programmed neutrophil death was PI3K-, p38 MAPK–, and JNK-dependent and evoked anti-inflammatory cytokines in macrophage cocultures. Neutrophils from patients with acute Crohn's disease, rheumatoid arthritis, or sepsis were susceptible to both IgA- and IVIG-mediated death. In contrast to IVIG, IgA did not promote cell death of quiescent neutrophils. Our findings suggest that plasma-derived IgA might provide a therapeutic option for the treatment of neutrophil-associated inflammatory disorders.

Influenza is a highly contagious, acute, febrile respiratory infection that can have fatal consequences particularly in individuals with chronic illnesses. Sporadic reports suggest that intravenous immunoglobulin (IVIg) may be efficacious in the influenza setting. We investigated the potential of human IVIg to ameliorate influenza infection in ferrets exposed to either the pandemic H1N1/09 virus (pH1N1) or highly pathogenic avian influenza (H5N1). IVIg administered at the time of influenza virus exposure led to a significant reduction in lung viral load following pH1N1 challenge. In the lethal H5N1 model, the majority of animals given IVIg survived challenge in a dose dependent manner. Protection was also afforded by purified F(ab′)2 but not Fc fragments derived from IVIg, supporting a specific antibody-mediated mechanism of protection. We conclude that pre-pandemic IVIg can modulate serious influenza infection-associated mortality and morbidity. IVIg could be useful prophylactically in the event of a pandemic to protect vulnerable population groups and in the critical care setting as a first stage intervention., Highlights • Intravenous immunoglobulin (IVIg), prepared prior to a pandemic, prevents pandemic influenza disease in ferrets. • IVIg effectively reduced viral levels of pandemic H1N1 influenza and prevented disease due to avian influenza H5N1. • This work has implications for preventing and treating pandemic influenza infections with IVIg before a vaccine is available. Influenza pandemics cause large numbers of infections and deaths. There is a lag between the identification of a pandemic and the development of vaccines. Future pandemics may be caused by influenza strains resistant to current anti-influenza drugs. New treatments are needed for future pandemic influenza outbreaks. We show that a readily available product (intravenous immunoglobuling – pooled antibodies from human donors) can prevent viral replication and disease caused by 2 strains of pandemic influenza viruses (“swine-flu” and “bird-flu”) in an appropriate animal model of influenza. This could form the basis of future treatments for severe influenza caused by pandemic strains.

Background Recurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates. Methods Distinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice. Results Characteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia. Conclusion Taken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection. Electronic supplementary material The online version of this article (10.1186/s12931-019-1057-3) contains supplementary material, which is available to authorized users.

International audience; Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflammatory components hemoglobin and heme, is a common event in the pathogenesis of numerous diseases with heterogeneous etiology and clinical features. A frequent adverse effect related to massive hemolysis is the renal injury and inflammation. Nevertheless, it is still unclear whether heme––a danger-associated molecular pattern––and ligand for TLR4 or upstream hemolysis-derived products are responsible for these effects. Well-characterized animal models of hemolysis with kidney impairment are needed to investigate how hemolysis drives kidney injury and to test novel therapeutic strategies. Here, we characterized the pathological processes leading to acute kidney injury and inflammation during massive intravascular hemolysis, using a mouse model of phenylhydrazine (PHZ)-triggered erythrocyte destruction. We observed profound changes in mRNA levels for markers of tubular damage (Kim-1, NGAL) and regeneration (indirect marker of tubular injury, Ki-67), and tissue and vascular inflammation (IL-6, E-selectin, P-selectin, ICAM-1) in kidneys of PHZ-treated mice, associated with ultrastructural signs of tubular injury. Moreover, mass spectrometry revealed presence of markers of tubular damage in urine, including meprin-α, cytoskeletal keratins, α-1-antitrypsin, and α-1-microglobulin. Signs of renal injury and inflammation rapidly resolved and the renal function was preserved, despite major changes in metabolic parameters of PHZ-injected animals. Mechanistically, renal alterations were largely heme-independent, since injection of free heme could not reproduce them, and scavenging heme with hemopexin in PHZ-administered mice could not prevent them. Reduced overall health status of the mice suggested multiorgan involvement. We detected amylasemia and amylasuria, two markers of acute pancreatitis. We also provide detailed characterization of renal manifestations associated with acute intravascular hemolysis, which may be mediated by hemolysis-derived products upstream of heme release. This analysis provides a platform for further investigations of hemolytic diseases and associated renal injury and the evaluation of novel therapeutic strategies that target intravascular hemolysis.

Objective Rheumatoid arthritis (RA), one of the most frequent chronic inflammatory rheumatic disorders, is characterized by the presence of autoantibodies and joint infiltration by activated immune cells, leading to cartilage and bone destruction. IgA occurs predominantly as monomers (mIgA) in plasma and regulates many cell responses through interaction with the Fcα receptor type I (FcαRI). FcαRI targeting by anti-FcαRI Fab inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine–based activation motif (ITAMi) configuration through SH2 domain–containing phosphatase 1 (SHP-1) recruitment. The aim of this study was to investigate the potential utility of mIgA for the treatment of arthritis by acting as an inducer of ITAMi signaling. Methods The effect of plasma-derived human mIgA on inhibition of multiple heterologous receptors was evaluated on FcαRI+ cell transfectants, blood phagocytes from healthy individuals, and synovial cells from RA patients. FcαRI-transgenic mice and wild-type mice treated with mIgA were studied in models of collagen antibody–induced arthritis (CAIA) and collagen-induced arthritis (CIA). The mice were assessed for development of arthritis using an arthritis score, and joint tissue samples were evaluated for the extent of leukocyte infiltration and expression of phosphatase. Results Treatment with mIgA impaired cell activation in an FcαRI–FcRγ-dependent manner, involving ITAMi signaling. Human mIgA or anti-FcαRI Fab were strongly effective in either preventing or attenuating CAIA or CIA in FcαRI-transgenic mice. Administration of mIgA markedly inhibited the recruitment of leukocytes to the inflamed joints of mice, which was associated with induction of SHP-1 phosphorylation in joint tissue cells. Moreover, mIgA reversed the state of inflammation in the synovial fluid of RA patients by inducing an ITAMi configuration. Conclusion These results demonstrate a therapeutic potential of human mIgA in experimental arthritis. The findings support future clinical exploration of mIgA for the treatment of RA.

Several mechanisms account for the beneficial effect of intravenous immunoglobulin (IVIg) in autoimmune and inflammatory diseases. These mechanisms include effects on the cellular compartment and on the humoral compartment. Thus, IVIg impacts on dendritic cells, macrophages, neutrophils, basophils, NK cells, and B and T lymphocytes. Several studies have emphasized that the antiinflammatory effect of IVIg is dependent on α2,6-sialylation of the N-linked glycan on asparagine-297 of the Fc portion of IgG. However, recent reports have questioned the necessity of sialylated Fc and the role of FcγRIIB in IVIg-mediated antiinflammatory effects. In view of the critical role played by Th17 cells in several autoimmune pathologies and the increasing use of IVIg in several of these conditions, by using neuraminidase-treated, desialylated IVIg, we addressed whether the α2,6-sialylation of IgG is essential for the beneficial effect of IVIg in experimental autoimmune encephalomyelitis (EAE), a Th17-driven condition, and for the reciprocal modulation of helper T-cell subsets. We observed no difference in the ability of IVIg to ameliorate EAE irrespective of its sialylation. Our findings thus show that sialylation of IVIg is not necessary for IVIg-mediated amelioration of EAE or for downregulation of Th17 cells and upregulation of regulatory T cells.

Immunoglobulin (Ig) replacement therapy has substantially changed the life of patients with primary antibody deficiency (PAD). In the majority of cases, patients with common variable immunodeficiency (CVID) or X-linked agammaglobulinaemia (XLA) now live to lead a near-normal life. Modern production facilities, a series of safety measures and a choice of several ways of administration make Ig replacement a safe and relatively easy therapy to use. The well-known presentations of PAD, such as pneumonia, septicaemia and other invasive bacterial infections 1, would continue to occur in PAD patients without regular replacement therapy. In this paper, we comment on the success and limitations of our present Ig replacement therapy in PAD. We also speculate how further improvement can be achieved in the treatment of complications from which PAD patients continue to suffer. IgG replacement effectively prevents pneumonia and invasive bacterial infections, as shown in several large cohorts. For instance, in a large Italian cohort of CVID patients, the prevalence of pneumonia was reduced from 49·0 to 20·5% upon initiation of Ig therapy 2. Prevention of pneumonia by Ig replacement therapy appears to be possible in a dose-dependent fashion. In a meta-analysis on IgG trough levels of 676 patients, the risk of pneumonia declined by 27% with each 0·1 g/kg body weight increment in the monthly IgG dose 3, although other factors, such as individual IgA levels, may determine the risk of pneumonia even more strongly 4. However, the effect of IgG replacement therapy on bacterial bronchitis and sinusitis in PAD patients is less clear. In the Italian CVID cohort, prevalence of chronic bacterial airway infections rose markedly from time at diagnosis through an observation period of a mean of 11 years of performed IgG replacement therapy. Frequency of both chronic bronchitis and sinusitis increased from 33·9 to 46·4% and from 36·6 to 54·0%, respectively 2. The increase of these conditions during Ig therapy was described similarly in XLA patients 1,4. Chronic bronchitis and sinusitis in PAD is due almost exclusively to chronic bacterial infection. The fierce, but ineffective inflammatory response, which inevitably follows the presence of bacteria in the sinus and lower airways, leads to repeated cycles of damage and repair of the airway epithelium. This process leads eventually to the formation of polyps and obstruction of the ostia of the paranasal sinuses, and to irreversible scarring and bronchiectasis. In bronchiectasis, airway clearance is permanently impaired, perpetuating the vicious cycle of infection and inflammation 5. Why is Ig replacement effective in preventing pneumonia, while markedly less so in preventing bacterial airway infection in PAD patients? The underlying reason may be that Ig replacement cannot fully substitute for an important part of the physiological airway defence. At the airway surface, the dominant isotype IgG is restricted to the alveolar space where it arrives after passive diffusion from the systemic circulation. Hence, inflammation in the alveolar space, i.e. pneumonia, is effectively prevented by systemic IgG replacement therapy. At the bronchial airway site, as well as in the nasal airways, however, IgA and IgM are the dominant isotypes in the immunocompetent individual. Both isotypes reach the airway lumen by active transport through the epithelium which is initiated by antibody-secreting cells located in the lamina propria of the airways 6. Patients with primary immunodeficiency (PID) frequently lack both these Ig isotypes and the related antibody-secreting cells. This renders them susceptible to bacterial and also viral airway infections. Viral infections in turn may predispose to bacterial infection by impairing mucociliary clearance 7, inducing phagocytic dysfunction 8 and/or promote bacterial adhesion 9. IgA at the luminal site is predominantly polymeric, which leads to differing immune functions in comparison to monomeric IgA. Monomeric IgA largely resembles IgG in triggering a proinflammatory response. Polymeric IgA more effectively immobilizes pathogens, prevents their adhesion or binds toxins 10. These mechanisms allow the removal of pathogens that are inhaled physiologically into the lower airways without causing inflammation, also referred to as immune exclusion 6. Why is IgA supposed to be an important part of the anti-bacterial airway defence in PAD patients, while apparently the vast majority of individuals with a selected IgA deficiency are not susceptible to prolonged bacterial or viral airway infection? The main reason is probably that, in CVID and XLA, patients also lack both IgA and IgM. IgM shares much of the immunological properties of polymeric IgA and may substitute for the lack of IgA in patients with selective IgA deficiency. IgA deficiency was the strongest independent risk factor for bronchiectasis in a prospective study with CVID and XLA patients 4. While it is widely accepted that Ig replacement therapy is not sufficiently effective in preventing airway disease, it is less clear which measures would ameliorate the disease course in the patients. The true prevalence of chronic sinusitis and bronchiectasis is still unknown. Currently, there is no consensus on the frequency on which these pathologies should be tested in routine care, how they should be tested or at what intervals. While chest CT and conventional chest X-ray are generally used to assess bronchiectasis, these techniques fail to detect a large proportion of bronchial pathologies. To date, there are no studies that demonstrate effective preventive or therapeutic measures against bronchiectasis in PAD patients. One of the major underlying reasons for the lack of studies is the difficulty to agree on a consensus protocol to reliably create quantitative data on bronchial pathology in a multi-centre setting. The international Chest CT in Antibody Deficiency Group (http://www.Chest-CT-Group.eu) aims to establish and validate a score for bronchiectasis and other structural lung disease for documenting the natural course of lung disease in PAD patients and potential effects in interventional studies. Preliminary data of the group show a steady increase of the prevalence of bronchiectasis with age from approximately 40% in patients aged less than 20 years to almost 80% in patients above 60 years in a large multi-national cohort of CVID patients. Assessing the prevalence and course of airway disease is only a prerequisite for improving the health of the patients. Which intervention is the most promising to improve efficacy over the present management? The role of antibiotic therapy has not been assessed thoroughly to date, and present practices range from no therapy to preventive antibiotic maintenance therapy. Different antibiotics may have differing effects which are not purely anti-bacterial, such as improvement of sputum rheology properties or anti-inflammatory effects, as shown for azithromycin in patients with cystic fibrosis 11. Hypertonic saline, which proved effective in improving sputum clearance in cystic fibrosis patients, may also be beneficial in PAD patients. Other measures, such as dornase alpha, nasal irrigation and physiotherapy, could also be effective, but have not yet been assessed formally. Most challenging, however, would be an effort to develop an Ig replacement strategy which is more physiological than the present practice. Is it feasible to replace serum IgA and IgM together with IgG systemically? In antibody-deficient patients, systemic replacement with serum IgA could lead potentially to the delivery of secretory IgA in the airway lumen, which is a natural process in healthy people. Indeed, these patients do not lack the expression of polymeric immunoglobulin receptor (pIgR), which is involved in the transepithelial transport of polymeric IgA and IgM (J-chain-positive IgA and IgM) on mucosal surfaces. However, this approach might not be as effective as desired for PAD patients, as serum IgA is mainly monomeric. It may eventually be more effective to apply Ig directly to the luminal site of the airways. Again, a number of challenges have to be met and are summarized in Table 1. Table 1 Outstanding questions for preventing airway infection in primary antibody deficiency In summary, there is still a long way to go until all issues of the care of PAD patients are resolved satisfactorily.

On March 15–17th, 2012 the 2nd international workshop on natural antibodies sponsored by CSL Behring AG took place in Bern, Switzerland. The focus of this workshop centered on new directions and in particular explored the “Future of Immunoglobulin Therapy”. Our international speakers addressed the major themes of understanding the origins of the immunoglobulin (Ig) repertoire and how we use it in our everyday lives to maintain homeostasis and fight infections and diseases. This Ig repertoire is already used as a therapeutic tool in the form of intravenous immunoglobulin (IVIG) for patients suffering from immunodeficiency and autoimmune disorders. The question now arises: what else can we learn and apply for the future of Ig therapy? Are there new directions, new ways of using the repertoire? These questions were discussed in sections covering modified Igs with modified efficacy including catalytic antibodies, the role of Fc receptors and complement inducing autoimmune mediated damage, Ig therapy beyond IgG in particular taking a look at the role of IgA and the therapeutic consequences of the different etiologies of excess amyloid beta in Alzheimer's disease and patients with Downs' syndrome. A major topic addressed the application of biomarkers to define new autoimmune diseases, to refine current therapies with a view to optimising the management of chronic diseases using both genetic and biomarker profiles to forewarn of possible future complications and to monitor fluctuations in disease status. Indeed, some of these topics push the frontiers of autoimmunity beyond its traditional scope and serve the purpose of making us re-evaluate the potential of using the Ig repertoire.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions including infection autoimmune diseases or immunodeficiency. Most immunoglobulin products recombinant or plasma derived are based on IgG antibodies whereas to date the use of IgA for therapeutic application has remained anecdotal. In particular purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work we sought to investigate whether polymeric IgA (plgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA like molecules. We found that ~15 of plasma plgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mlgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with plgA and with similar efficacy as colostrum derived SC. In comparison with plgA the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma derived IgM. In vitro plasma derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen resulting in a delay of bacteria induced damage targeted to polarized Caco 2 cell monolayers. The sum of these novel data demonstrates that association of plasma derived IgA or IgM with recombinant/colostrum derived SC is feasible and yields SIgA and SIgM like molecules with similar biochemical and functional characteristics as mucosa derived immunoglobulins. © 2013 by The American Society for Biochemistry and Molecular Biology Inc.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data.

BACKGROUND: Sialylation of the N-linked glycan on asparagine-297 within the Fc region of intravenous gammaglobulins (IVIgs) was shown to be necessary for efficacy of IVIg in the amelioration of experimental inflammatory arthritis. To test the role for Fc sialylation of IVIg in immune modulating therapies beyond the K/BxN arthritis model, we examined the efficacy of sialylated compared to nonsialylated IVIg for the ability to attenuate immune thrombocytopenia (ITP) in a mouse model that approximates the clinical setting of human ITP. STUDY DESIGN AND METHODS: We used a published, passive anti-platelet (PLT) dose-escalation mouse model of ITP that approximates clinical ITP. PLT counts were followed over time before and after IVIg treatment. IVIg from two different manufacturers was used to prepare untreated and neuraminidase-treated IVIg. Sambucus nigra agglutinin (SNA) affinity chromatography was used to obtain sialic acid–enriched and -depleted IVIg. Sialic acid content was determined using Western blot, enzyme-linked immunosorbent assay, and high-performance liquid chromatography. RESULTS: Results were the same using sialylated and desialylated (neuraminidase-treated) IVIg from two different manufacturers. No differences were observed between sialic acid–enriched and -depleted IVIg compared to normal IVIg in their efficacy to alleviate ITP. Using quantitative reverse transcription–polymerase chain reaction, no increase in the spleen FcγRIIB mRNA was detectable, but a pronounced increase of FcγRIIB mRNA in the marrow was seen after IVIg administration. CONCLUSIONS: We conclude that IVIg ameliorates experimental ITP by a mechanism that is independent of sialylation either in the Fc or the Fab region of IVIg.

Wnt/β-catenin signaling controls multiple steps of neural crest development, ranging from neural crest induction, lineage decisions, to differentiation. In mice, conditional β-catenin inactivation in premigratory neural crest cells abolishes both sensory neuron and melanocyte formation. Intriguingly, the generation of melanocytes is also prevented by activation of β-catenin in the premigratory neural crest, which promotes sensory neurogenesis at the expense of other neural crest derivatives. This raises the question of how Wnt/β-catenin signaling regulates the formation of distinct lineages from the neural crest. Using various Cre lines to conditionally activate β-catenin in neural crest cells at different developmental stages, we show that neural crest cell fate decisions in vivo are subject to temporal control by Wnt/β-catenin. Unlike in premigratory neural crest, β-catenin activation in migratory neural crest cells promotes the formation of ectopic melanoblasts, while the production of most other lineages is suppressed. Ectopic melanoblasts emerge at sites of neural crest target structures and in many tissues usually devoid of neural crest-derived cells. β-catenin activation at later stages in glial progenitors or in melanoblasts does not lead to surplus melanoblasts, indicating a narrow time window of Wnt/β-catenin responsiveness during neural crest cell migration. Thus, neural crest cells appear to be multipotent in vivo both before and after emigration from the neural tube but adapt their response to extracellular signals in a temporally controlled manner.

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Intravenous immunoglobulin (IVIG) preparations are increasingly used for the treatment of autoimmune and chronic inflammatory diseases. Naturally occurring autoantibodies against Siglec-9 and Fas are thought to contribute to the anti-inflammatory effects of IVIG via cell death regulation of leukocytes and tissue cells. Dimeric IVIG fractions are suspected to contain idiotypic (Id)-anti-idiotypic complexes of antibodies, which might also include anti-Siglec-9 and anti-Fas autoantibodies.

Auto-reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self-antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self-antigens is not robust. To investigate the fate of nervous system-specific CD8(+) T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus-derived CD8(+) T-cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8(+) T-cell epitopes induced robust tolerance of endogenous auto-reactive T cells, which proved thymus-independent and was mediated by cross-presenting bone-marrow-derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia-derived antigens in DC, suggesting that peripheral tolerance of CD8(+) T cells results from uptake and presentation by steady state DC.

SummaryHigh-dose intravenous immunoglobulin (IVIg) preparations are used currently for the treatment of autoimmune or inflammatory diseases. Despite numerous studies demonstrating efficacy, the precise mode of action of IVIg remains unclear. Paradoxically, IgG can exert both pro- and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low-dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific receptors (FcγR) on innate immune effector cells. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. How this anti-inflammatory effect is mediated has not yet been elucidated fully, and several mutually non-exclusive mechanisms have been proposed. This paper represents the proceedings of a session entitled ‘IVIg – Understanding properties and mechanisms’ at the 6th International Immunoglobulin Symposium that was held in Interlaken on 26–28 March 2009. The presentations addressed how IgG may affect the cellular compartment, evidence for IVIg-mediated scavenging of complement fragments, the role of the dimeric fraction of IVIg, the anti-inflammatory properties of the minor fraction of sialylated IgG molecules, and the genetic organization and variation in FcγRs. These findings demonstrate the considerable progress that has been made in understanding the mechanisms of action of IVIgs, and may influence future perspectives in the field of Ig therapy.

Inhibitory anti-muscarinic receptor type 3 (M3R) antibodies may contribute to the pathogenesis of Sjögren's syndrome (SS), and putative anti-M3R blocking antibodies in intravenous immunoglobulin (IVIg) have been suggested as a rationale for treatment with IVIg. We investigated the presence of subtype-specific anti-MR autoantibodies in healthy donor and SS sera using MR-transfected whole-cell binding assays as well as M1R and M3R peptide ELISAs. Control antibodies against the second extracellular loop of the M3R, a suggested target epitope, were induced in rabbits and found to be cross-reactive on the peptides M3R and M1R. The rabbit antibodies had neither an agonistic nor an antagonistic effect on M3R-dependent ERK1/2 signalling. Only one primary SS (out of 5 primary SS, 2 secondary SS and 5 control sera) reacted strongly with M3R transfected cells. The same SS serum also reacted strongly with M1R and M2R transfectants, as well as M1R and two different M3R peptides. Strong binding to M1R and low-level activities against M3R peptides were observed both in SS and control sera. IVIg showed a strong reactivity against all three peptides, especially M1R. Our results indicate that certain SS individuals may have antibodies against M1R, M2R and M3R. Our results also suggest that neither the linear M3R peptide nor M3R transfectants represent suitable tools for discrimination of pathogenic from natural autoantibodies in SS.

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

The CD27/CD70-interaction has been shown to provide a costimulatory and survival signal for T cells in vitro and in vivo. Recently, CD70 expression by DC was found to be important for the priming of CD8+ T cells. We show here that blocking CD70 interactions has a significant impact on priming of CD8+ T cell responses by vaccinia virus (VV), Listeria monocytogenes and vesicular stomatitis virus (VSV) in mice. However, the priming of specific CD8+ T cells upon infection with lymphocytic choriomeningitis virus (LCMV) was only marginally reduced by CD70-blockade. Blocking of CD70 prevented CD8+ T cell priming in DIETER mice, a model in which presentation of LCMV-derived epitopes can be induced selectively in dendritic cells (DC). In contrast, CD70-CD27 interactions were not important for the priming of VSV-specific CD4+ T cells or class switch of neutralizing antibodies. As we show that priming of CD8+ T cells by the pathogens used here is dependent on antigen presentation by DC and that infection results in up-regulation of CD70 on DC, we conclude that CD70 expression on DC plays an important role in the priming of CD8+ T cells by pathogens. Moreover, the lack of CD70 cannot be completely compensated for by other costimulatory molecules.

We have recently shown that the majority of allergens can be represented by allergen motifs. This observation prompted us to experimentally investigate the synthesized peptides corresponding to the in silico motifs with regard to potential IgE binding and cross-reactions with allergens. Two motifs were selected as examples to conduct in vitro studies. From the first motif, derived from allergenic MnSOD sequences, the motif stretch of the allergen Asp f 6 was selected and synthesized as a peptide (MnSOD Mot). The corresponding full-length MnSOD was also expressed in Escherichia coli and both were compared for IgE reactivity with sera of patients reacting to the MnSOD of Aspergillus fumigatus or Malassezia sympodialis. For the second motif, the invertebrate tropomyosin sequences were aligned and a motif consensus sequence was expressed as a recombinant protein (Trop Mot). The IgE reactivity of Trop Mot was analyzed in ELISA and compared to that of recombinant tropomyosin from the shrimp Penaeus aztecus (rPen a 1) in ImmunoCAP. MnSOD Mot was weakly recognized by some of the tested sera, suggesting that the IgE binding epitopes of a multimeric globular protein such as MnSOD cannot be fully represented by a motif peptide. In contrast, the motif Trop Mot showed the same IgE reactivity as shrimp full-length tropomyosin, indicating that the major allergenic reactivity of a repetitive structure such as tropomyosin can be covered by a motif peptide. Our results suggest that the motif-generating algorithm may be used for identifying major IgE binding structures of coiled-coil proteins.

Despite the paradigm that carbohydrates are T cell-independent antigens, isotype-switched glycan-specific immunoglobulin G (IgG) antibodies and polysaccharide-specific T cells are found in humans. We used a systems-level approach combined with glycan array technology to decipher the repertoire of carbohydrate-specific IgG antibodies in intravenous and subcutaneous immunoglobulin preparations. A strikingly universal architecture of this repertoire with modular organization among different donor populations revealed an association between immunogenicity or tolerance and particular structural features of glycans. Antibodies were identified with specificity not only for microbial antigens but also for a broad spectrum of host glycans that serve as attachment sites for viral and bacterial pathogens and/or exotoxins. Tumor-associated carbohydrate antigens were differentially detected by IgG antibodies, whereas non-IgG2 reactivity was predominantly absent. Our study highlights the power of systems biology approaches to analyze immune responses and reveals potential glycan antigen determinants that are relevant to vaccine design, diagnostic assays, and antibody-based therapies.

Intravenous immunoglobulin (IVIG) is involved in many complex mechanisms that act in synergy including expression and function of Fc receptors, complement activation, the cytokine network, interaction with the anti-idiotypic network and modulation of B and T cell activation. To gain insight into the early effects of IVIG on this broad range of activities at the gene level we performed DNA microarray analysis. Human whole blood was incubated in vitro for 4 h followed by extraction of RNA which was hybridized to a chip containing 8793 genes. About 75 upregulated genes and 21 downregulated genes were identified using a cut off for the false discovery rate of 5%. These genes are associated with a wide range of cellular immune functions in line with the broad mechanism of action of IVIG. A striking upregulation of a series of genes coding for chemokines was measured. This finding was confirmed at the protein level as pharmacologically relevant concentrations of CXCL9 and CXCL10 were measured in serum. Interestingly, IVIG shows a partial overlap of its gene expression program with lipopolysaccharide. Our data suggests multiple hypotheses regarding the pharmacology of IVIG that must be validated by complementary studies.

FcαRI (CD89), the human Fc receptor for IgA, is highly expressed on neutrophil granulocytes. In this study, we show that FcαRI induces different forms of neutrophil death, depending on the inflammatory microenvironment. The susceptibility of inflammatory neutrophils from sepsis or rheumatoid arthritis toward death induced by specific mAb, or soluble IgA at high concentrations, was enhanced. Although unstimulated cells experienced apoptosis following anti-FcαRI mAb stimulation, preactivation with cytokines or TLR agonists in vitro enhanced FcαRI-mediated death by additional recruitment of caspase-independent pathways, but this required PI3K class IA and MAPK signaling. Transmission electron microscopy of FcαRI-stimulated cells revealed cytoplasmic changes with vacuolization and mitochondrial swelling, nuclear condensation, and sustained plasma membrane. Coculture experiments with macrophages revealed anti-inflammatory effects of the partially caspase-independent death of primed cells following FcαRI engagement. Our data suggest that FcαRI has the ability to regulate neutrophil viability and to induce different forms of neutrophils depending on the inflammatory microenvironment and specific characteristics of the ligand–receptor interactions. Furthermore, these findings have potential implications for FcαRI-targeted strategies to treat neutrophil-associated inflammatory diseases.

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the family of programmed cell death-inducing cytokines. Apo2L/TRAIL induces apoptosis in a wide variety of tumor cells. Tumor cells that are resistant to Apo2L/TRAIL-induced apoptosis can be sensitized by chemotherapeutic drugs and other agents via an unknown mechanism. Here we report that PG490 (triptolide), a diterpene triepoxide extracted from the Chinese herb Tripterygium wilfordii and used in traditional Chinese medicine, sensitizes lung cancer but not normal human bronchial epithelial cells to Apo2L/TRAIL-induced apoptosis. Sensitization was accompanied by caspase-3 and caspase-8 activation, whereas no cleavage of caspase-9 was observed. Determination of cell surface receptors by flow cytometry demonstrated no difference in Apo2L/TRAIL-R1 and -R2 expression, the two receptors with functional death domains, between resistant and sensitized cells. In cells treated with the combination of Apo2L/TRAIL and PG490, we observed activation of ERK2, a member of the mitogen-activated protein kinase family. Furthermore, sensitization could be blocked by the ERK inhibitor U0126 but not the p38 inhibitor SB203580, suggesting that activation of ERK2 is required for this effect. In addition, sensitization of lung cancer cells was also seen in ex vivo culture of lung cancer tissue from four patients who underwent surgery. Immunohistochemical staining showed a clear reduction in proliferation cell nuclear antigen (PCNA) in tissue treated with Apo2L/TRAIL and PG490. In conclusion, apoptosis induced by the combination of Apo2L/TRAIL and PG490 warrants further evaluation as a potential new strategy for the treatment of lung cancer.

Anti-idiotypic antibodies (anti-Id) have been described against idiotypes expressed on various autoantibodies. Since an immunoregulatory effect has been postulated for anti-Id, modulation of the anti-Id response in autoimmune disease may be of interest. In chronic immune thrombocytopenic purpura (AITP), autoantibodies directed mainly against platelet membrane glycoprotein (GP) IIb/IIIa cause platelet destruction by Fc-mediated phagocytosis or by complement lysis. We have previously reported on the generation of two recombinant anti-GPIIb/IIIa autoantibody fragments (PDG-X, PDG-B), that are specific for conformationally intact GPIIb/IIIa and inhibit binding of autoantibodies from patients with AITP. In the present study, we show that anti-GPIIb/IIIa specificities are not limited to a single individual by isolating five additional anti-GPIIb/IIIa autoantibody fragments from a second phagemid Fab library of an unrelated healthy donor. Using soluble Fab of PDG-X and PDG-B as antigens for panning Fab phagemid libraries from healthy human individuals, we isolated anti-Id phage clones specific for PDG-X or PDG-B. In addition they inhibited the binding of PDG-X or PDG-B to GPIIb/IIIa. Amino acid sequence comparison between these specific antiId and GPIIb/IIIa was performed. Generation of these anti-Id directed against pathologically relevant anti-GPIIb/IIIa autoantibodies may represent a new suitable and specific therapeutic option for the treatment of antibody-mediated AITP.

IVIG modulates T cell activation in vitro and inflammatory-autoimmune conditions in vivo. Sialylation of IgG, Fc receptor interactions, modulation of monocyte/macrophage/B cell functions have been implicated in IVIG effects. Subcutaneous IgG (SCIG) therapy is increasingly used for IgG replacement but whether these preparations share the effects of IVIG on T cell modulation is not documented. We compared the potency of SCIG-Hizentra™ (20% IgG preparation) with IVIG-Privigen® (10% IgG) for T cell inhibition, and assessed the involvement of IgG sialylation, monocytes and B cells in this process. Human PBMCs or sorted cells were cultured 3-7 days, and T cells were stimulated with immobilized anti-CD3 mAb or Candida antigen. Thymidine incorporation into DNA was quantitated and cytokines assayed by ELISA/Luminex® assay. IVIG and SCIG both dose-dependently (1-20mg/ml) inhibited (up to >80%) T cell proliferation to anti-CD3 mAb. Response to Candida albicans was comparably inhibited by IVIG and SCIG by 50-80% at 10mg/ml with inhibition even at 3mg/ml (P

Background and Objectives To evaluate standard intravenous immunoglobulin (IVIG) as an alternative to intravenous cytomegalovirus hyperimmune immunoglobulin (CMVIG) for prophylaxis and therapy of cytomegalovirus (CMV) disease, we measured the ELISA and neutralizing titres of CMV-specific antibodies in CMVIG and IVIG preparations. Materials and Methods Anti-CMV-IgG ELISA and neutralizing titres (fibroblast-based test) in CMVIG CG (Cytogam®, n = 20), CMVIG CT (Cytotect® CP, n = 3), IVIG P (Privigen®, n = 32) and IVIG K/G (Kiovig®/Gammagard®, n = 5) were compared, and IgG subclasses 1–4 were determined by nephelometry. Results Cytomegalovirus hyperimmune immunoglobulins contained more than fourfold higher CMV ELISA and CMV-neutralizing activity per gram of IgG than the standard IVIGs. Pooled data for all four products showed a significant correlation between anti-CMV-IgG ELISA and neutralizing titres (r = 0·93, P

Intravenous administration of polyclonal and monoclonal antibodies has proven to be a clinically valid approach in the treatment or at least relief of many acute and chronic pathologies such as infection immunodeficiency and a broad range of autoimmune conditions. Plasma derived IgG or recombinant IgG are most frequently used for intravenous or subcutaneous administration whereas a few IgM based products are available as well.Wehave established recently that secretory like IgA and IgM can be produced upon association of plasma derived polymeric IgA and IgM with a recombinant secretory component. As a next step toward potential future mucosal administration we sought to unravel the mechanisms by which these secretory Igs protect epithelial cells located at the interface between the environment and the inside of the body. By using polarized epithelial Caco 2 cell monolayers and Shigella flexneri as a model enteropathogen we found that polyspecific plasma derived SIgA and SIgM fulfill many protective functions including dose dependent recognition of the antigen via formation of aggregated immune complexes reduction of bacterial infectivity maintenance of epithelial cell integrity and inhibition of proinflammatory cytokine/chemokine production by epithelial cells. In this in vitro model devoid of other cellular or molecular interfering partners IgM and secretory IgM showed stronger bacterial neutralization than secretory IgA. Together these data suggest that mucosally delivered antibody preparations may be most effective when combining both secretory like IgA and IgM which together play a crucial role in preserving several levels of epithelial cell integrity. © 2014 by The American Society for Biochemistry and Molecular Biology Inc.

High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN–expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1–2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab′)2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.

The role of autoantibodies against the alpha-subunit of the human high-affinity IgE receptor (FcepsilonRIalpha) in the pathogenesis of chronic idiopathic urticaria (CIU) is controversial. We have shown that these antibodies are widespread, apparently non-pathogenic and belong to the natural antibody repertoire. To clarify this controversy, we constructed antibody libraries from both healthy donors and CIU patients with active disease. Here we describe the first three high affinity IgM anti-FcepsilonRIalphaautoantibodies isolated from normal and urticaria libraries. Sequence analysis revealed germline VH in both cases paired with a slightly mutated VL, thus supporting their classification as natural antibodies. Strikingly, one major IgM clone was present in both CIU patients and normal donors. The anti-FcepsilonRIalpha autoantibodies recognize FcepsilonRIalpha on cells, but are non-anaphylactogenic on blood basophils, except when IgE is removed from the receptor. Based on their functional activities we propose a concept of "conditional autoimmunity" where natural anti-FcepsilonRIalphaautoantibodies can become pathogenic dependent on the state of occupancy of the FcepsilonRIalpha by its natural ligand IgE.

Natural antibodies provide an early defense mechanism against pathogens, show a frequent self-reactivity, and are present throughout life. Two questions concern the physiological control of self-reactivity and the pathogenetic link to autoimmune disease. Here we propose a concept of conditional autoimmunity involving natural antibodies against the alpha chain of the high-affinity receptor for IgE (Fc(epsilon)RIalpha ). Like other natural antibodies, anti-Fc(epsilon)RIalpha antibodies are found in sera of healthy donors. We now report the first human recombinant anti-Fc(epsilon)RIalpha autoantibodies isolated by repertoire cloning from a human tonsillar IgM library. These high-affinity antibodies recognize Fc(epsilon)RIalpha on cells and trigger histamine release from freshly isolated blood basophils. However, the latter effect requires IgE removal from the Fc(epsilon)RI. The same conditional histamine release is seen when using sera from individual normal donors and affinity-purified anti-Fc(epsilon)RIalpha antibodies isolated from multidonor therapeutic IgG preparations. We propose that such anti-Fc(epsilon)RIalpha antibodies can become pathogenic and that this is dependent on the state of occupancy of the Fc(epsilon)RIalpha by its natural ligand IgE. We suggest that an imbalance between Fc(epsilon)RIalpha occupancy and natural anti-Fc(epsilon)RIalpha antibodies may be implicated in the pathogenesis of autoimmune urticaria.

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcεRI) on mast cells and basophils. Therefore, the IgE/FcεRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcεRI, and interferes with binding of IgE to FcεRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cε3 and one within the Cε4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cε3 region predominantly blocks binding of IgE to FcεRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cε4 region may explain how BSW17 recognizes FcεRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti–allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

According to Jerne’s network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunisation, and confirm Jerne’s hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.

An essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. As bacteriophages are adapted to this environment we used epitope-displaying M13 bacteriophages as carriers for an experimental oral anti-IgE vaccine. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti-idiotypic Fab of the non-anaphylactogenic monoclonal anti-IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein were still recognized by BSW17 whereas pIII-expressed epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti-IgE antibodies. In contrast, oral immunization with the purified, pVIII protein displaying the mimotope induced anti-phage but no anti-IgE antibodies. After feeding a single dose of mimotope-displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. Our results show that epitope-displaying bacteriophages can be used to induce an epitope-specific antibody response via the oral route.

Antigen-specific human IgE is in short supply. Thus, we sought to determine the yet unknown specificity of a widely available human IgE, namely the myeloma cell line U266-derived IgE-ND. For this purpose highly specific peptides able to mimic the putative antigen recognized by IgE-ND were isolated from phage-display random peptide libraries. Interestingly, we found linear sequence homologies of the IgE-ND-binding peptides with self antigens and a xenoantigen from Thiobacillus ferrooxidans. However, none of these antigens was recognized by IgE-ND. Nevertheless, our approach may be applied to identify antigen specificities of myeloma antibodies. Importantly, the mimotopes were anaphylactogenic in a histamine release assay using human basophils sensitized with IgE-ND. Thus, our mimotopes represent functional albeit synthetic antigens and may be used to study human antigen-specific IgE responses.

Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto-Ab) can be either pathogenic or non-pathogenic. Auto-Ab reacting with the α-subunit of the high-affinity receptor for IgE (FcϵRIα) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti-FcϵRIα auto-Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity-purified the anti-FcϵRIα Ab from an IVIg preparation using recombinant FcϵRIα. Interestingly, these anti-FcϵRIα auto-Ab showed no evidence of histamine release but strongly cross-reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcϵRIα. Since the cross-reacting Ab are non-anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti-FcϵRIα auto-Ab belong to the natural Ab and serve as the parental Ab for some anti-TTd Ab.

Autoimmune thrombocytopenic purpura (AITP) is caused by autoantibodies predominantly against platelet membrane glycoproteins (GP) IIb/IIIa and GPIb/IX. Naturally occurring autoantibodies have been described against a variety of autoantigens; it has been suggested that perturbation of their regulation may be associated with autoimmune diseases. Using a combinatorial Fab phagemid library from an individual immunized with human RhD+ red blood cells, we evaluated the presence of natural anti-GPIIb/IIIa autoantibodies as well as their relation to AITP-associated anti-GPIIb/IIIa autoantibodies. Selection on native GPIIb/IIIa and characterization of positive clones by inhibition studies against murine monoclonal anti-GPIIb/IIIa antibodies and by DNA analysis revealed the presence of two distinct recombinant anti-GPIIb/IIIa autoantibodies, which partially inhibited binding of affinity-purified platelet-associated autoantibodies from 8/12 AITP patients. Our results demonstrated that GPIIb/IIIa-specific Fab directed against conformational epitopes within the GPIIb/IIIa complex may be cloned from the genome of an individual immunized with RhD+ red blood cells, who was not affected by AITP. The partial inhibition of binding of platelet-associated autoantibodies from AITP patients to GPIIb/IIIa by the recombinant anti-GPIIb/IIIa phage clones suggests recognition of closely related antigenic epitopes. These phage clones may represent down-regulated, potentially pathological autoantibodies and could be used as new tools for investigation of AITP.

It is well established that heat–denatured IgE is no longer capable of binding to FcεRI. We have found an antibody that interacts with heat–denatured IgE. Interestingly, this antibody can also be used to detect some serum IgE, but not IgE synthesized de novo in vitro. However, native IgE can be transformed into an IgE that is recognized by this antibody, if antigen is added. Our data indicate that physiological mechanisms exist that biologically inactivate IgE which might still be mistaken for 'functional' IgE by assays based on polyclonal antibodies.

Although microvasculitis is one of the more common manifestations of systemic lupus erythematosus, there is no data on the hemodynamics of the skin microcirculation in such patients. The combination of dynamic capillaroscopy measuring capillary blood cell velocity (CBV) and laser Doppler fluxmetry (LDF) was used to simultaneously evaluate the nutritional and the total skin microcirculation in the fingers of 24 consecutive patients with SLE, using normal matched subjects as controls. The nutritional skin flow, as assessed by the CBV, was significantly impaired in patients, as compared with controls, both at rest (p = 0.001) and during postocclusive reactive hyperemia (p = 0.006). By contrast, no differences were observed in total skin microcirculation, as assessed by LDF (n.s.). There was no significant correlation between hemodynamic parameters and the presence of Raynaud's phenomenon, morphological capillary changes or anticardiolipine antibodies. There was no correlation between the magnitude of the alteration of the capillary blood flow and capillary morphological abnormalities, suggesting that the microvascular damage might be caused by different pathophysiological mechanisms.

BACKGROUND Ischemia/reperfusion injury of lower extremities and associated lung damage may result from thrombotic occlusion, embolism, trauma, or surgical intervention with prolonged ischemia and subsequent restoration of blood flow. This clinical entity is characterized by high morbidity and mortality. Deprivation of blood supply leads to molecular and structural changes in the affected tissue. Upon reperfusion inflammatory cascades are activated causing tissue injury. We therefore tested preoperative treatment for prevention of reperfusion injury by using C1 esterase inhibitor (C1 INH). METHODS AND FINDINGS Wistar rats systemically pretreated with C1 INH (n = 6), APT070 (a membrane-targeted myristoylated peptidyl construct derived from human complement receptor 1, n = 4), vehicle (n = 7), or NaCl (n = 8) were subjected to 3h hind limb ischemia and 24h reperfusion. The femoral artery was clamped and a tourniquet placed under maintenance of a venous return. C1 INH treated rats showed significantly less edema in muscle (P

Eric Mohler, Arthur Nikkel, Richard Radek, Holly Robb, Michael Schrimpf, Jeffrey Waring, Chih-Hung Lee, Murali Gopalakrishnan, AbbVie Inc., North Chicago, Illinois, United States; AbbVie, North Chicago, Illinois, United States; AbbVie Inc., North Chicago, Illinois, United States; AbbVie Inc., North Chicago, Illinois, United States; AbbVie, North Chicago, Illinois, United States. Contact e-mail: robert.s.bitner@abbvie.com

Objective Plasmacytoid dendritic cells (PDCs) produce high concentrations of interferon-α (IFNα) following exposure to immune complexes containing nucleic acids. We previously reported that serum from healthy donors inhibits IFNα production by PDCs in response to systemic lupus erythematosus (SLE) immune complexes, and that inhibition is mediated, in part, by IgG. IgG is the major component of intravenous immunoglobulin and is well known to exert antiinflammatory properties. Although suppression of inflammation by the sialylated subfraction of IgG has been implicated in some models, the mechanism of IFNα inhibition by IgG and the importance of sialylation have not been studied. Methods SLE immune complexes or synthetic Toll-like receptor (TLR) agonists were used to stimulate total or individual cell–depleted human mononuclear cell cultures in the presence or absence of IgG, Fc fragments, F(ab′)2 fragments, and their sialylated or unsialylated subfractions. Cytokines were quantified by enzyme-linked immunosorbent assay. Results We identified 2 distinct mechanisms by which IgG inhibits IFNα production. First, IgG Fc fragments inhibited SLE immune complex–stimulated IFNα production via a sialic acid–independent mechanism, by inhibiting immune complex binding to Fcγ receptor IIa on PDCs. In contrast, the F(ab′)2 fragment of the sialylation-enriched fraction of IgG inhibited TLR-7 or TLR-9 agonist–induced IFNα production but did not require the sialic acid residue itself. The inhibitory activity of IgG on TLR agonist–induced IFNα required monocyte production of prostaglandin E2, a potent suppressor of IFNα production by PDCs. Conclusion IgG attenuates IFNα production by PDCs by both cell surface receptor and intracellular pathways, depending on the nature of the inducing stimulus.

Intravenous Immunoglobulin (IVIG) has been proposed as a potential therapeutic for Alzheimer's disease (AD) and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aβ) antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aβ -specific antibodies (pAbs-Aβ) on aggregation, toxicity and phagocytosis of Aβ in vitro, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aβ specifically bound to Aβ and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aβ inhibited Aβ-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aβ binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aβ also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aβ by BV-2 microglia. Phagocytosis of Aβ depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aβ-mediated LTP inhibition in mouse hippocampal slices. Therefore, our in vitro results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.

An anti-inflammatory effect of reconstituted High Density Lipoprotein (rHDL) has been demonstrated in atherosclerosis and in sepsis models. An increase of adhesion molecules as well as tissue factor expression on endothelial cells in response to inflammatory or danger signals are attenuated by the treatment with rHDL. Here we show the inhibitory effect of rHDL on the activation of human leukocytes in a whole blood assay as well as on monocyte-derived human dendritic cells (DC). Multiplex analysis of human whole blood showed that phytohaemagglutinin (PHA)-induced secretion of the cytokines IL-1β, IL-1RA, IL-2R, IL-6, IL-7, IL-12(p40), IL-15 and IFN-α was inhibited. Furthermore, an inhibitory effect on the production of the chemokines CCL-2, CCL-4, CCL-5, CXCL-9 and CXCL-10 was observed. Activation of granulocytes and CD14+ monocytes by PHA is inhibited dose-dependently by rHDL shown as decreased up-regulation of ICAM-1 surface expression. In addition, we found a strong inhibitory effect of rHDL on toll-like receptor 2 (TLR2)- and TLR4-mediated maturation of DC. Treatment of DC with rHDL prevented the up-regulation of cell surface molecules CD80, CD83 and CD86 and it inhibited the TLR-driven activation of inflammatory transcription factor NF-κB. These findings suggest that rHDL prevents activation of crucial cellular players of cellular immunity and could therefore be a useful reagent to impede inflammation as well as the link between innate and adaptive immunity.

Anti-IgE mAbs have always been used to trigger mediator release from basophils or mast cells. Now nonanaphylactogenic anti-human IgE Abs are in clinical evaluation as a therapeutic agent against atopic disease. We have found a mAb that is nonanaphylactogenic but recognizes receptor-bound IgE. Interestingly, the Ab prevents the association of IgE with its receptor if immune complexes were formed between IgE and anti-IgE mAb. This explained the phenomenon that addition of anti-IgE Ab to receptor-bound IgE resulted in a decrease of receptor-bound IgE, because IgE dissociating from the receptor was complexed, altering the thermodynamic balance of receptor-bound vs free IgE. Our data show that there are nonanaphylactogenic Abs that do not directly interfere with the receptor binding site on IgE and are capable of preventing the association of IgE with its high affinity receptor. This unique feature would render such an anti-IgE Ab a possible candidate for immunotherapy of atopy.