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Recently, numerous studies report bats as reservoirs of emerging pathogens with little to no signs of infections. This is thought to be connected to the unique immune system of bats, which remains poorly characterized. Despite the physiological importance of the Neonatal Fc receptor (FcRn) in the homeostasis of IgG antibodies, it is unclear how its functional activity is evolutionary conservative among mammals, and so is the case for bats. Using surface plasmon resonance-based technology, we tested the interactions of IgG antibodies isolated from three bat species with recombinant human and mouse FcRn. Our data show that IgG from the studied bat species binds to both human and mouse FcRn, albeit with distinct affinities. Importantly, the binding pattern of bat IgG is similar to human IgG. This confirms the conservative nature of IgG-FcRn interaction and highlights the importance of FcRn IgG salvaging system in bats. • Neonatal Fc receptor (FcRn) plays important role of homeostasis of IgG in different species. • Recombinant FcRn binds IgG from three different bat species. • Human FcRn binds bat IgG with similar quantitative parameters as human IgG. • Bat IgG might be recycled and transferred from mother to foetus in a same way as other species IgG. [ABSTRACT FROM AUTHOR]

Hemolytic diseases such as sickle-cell disease, β-thalassemia, malaria, and autoimmune hemolytic anemia continue to present serious clinical hurdles. In these diseases, lysis of erythrocytes causes the release of hemoglobin and heme into plasma. Extracellular heme has strong proinflammatory potential and activates immune cells and endothelium, thus contributing to disease pathogenesis. Recent studies have revealed that heme can interfere with the function of plasma effector systems such as the coagulation and complement cascades, in addition to the activity of immunoglobulins. Any perturbation in such functions may have severe pathological consequences. In this review we analyze heme interactions with coagulation, complement, and immunoglobulins. Deciphering such interactions to better understand the complex pathogenesis of hemolytic diseases is pivotal. [ABSTRACT FROM AUTHOR]

• Human serum IgA is able to interact with novel antigens after contact with heme. • After interaction with Fe(II) ions human serum IgA become polyspecific. • Human serum IgA shows increased immunoreactivity after exposure to low pH buffers. Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity. [ABSTRACT FROM AUTHOR]

The bat immune system features multiple unique properties such as dampened inflammatory responses and increased tissue protection, explaining their long lifespan and tolerance to viral infections. Here, we demonstrated that body temperature fluctuations corresponding to different physiological states in bats exert a large impact on their antibody repertoires. At elevated temperatures typical for flight, IgG from the bat species Myotis myotis and Nyctalus noctula show elevated antigen binding strength and diversity, recognizing both pathogen-derived antigens and autoantigens. The opposite is observed at temperatures reflecting inactive physiological states. IgG antibodies of human and other mammals, or antibodies of birds do not appear to behave in a similar way. Importantly, diversification of bat antibody specificities results in preferential recognition of damaged endothelial and epithelial cells, indicating an anti-inflammatory function. The temperature-sensitivity of bat antibodies is mediated by the variable regions of immunoglobulin molecules. Additionally, we uncover specific molecular features of bat IgG, such as low thermodynamic stability and implication of hydrophobic interactions in antigen binding as well as high prevalence of polyreactivity. Overall, our results extend the understanding of bat tolerance to disease and inflammation and highlight the link between metabolism and immunity., (© 2024. The Author(s).)

Background: Free labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis., Objectives: To investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis., Methods: The effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively. Its effect on ferric-chloride (FeCl 3 )-induced arterial thrombosis and lung perfusion following hemin injection was assessed in wild-type mice., Results: Erythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride-induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi., Conclusion: Hydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antibodies play a pivotal role in protecting from SARS-CoV-2 infection, but their efficacy is challenged by the continuous emergence of viral variants. In this study, we describe two broadly neutralizing antibodies cloned from the memory B cells of a single convalescent individual after infection with ancestral SARS-CoV-2. Cv2.3194, a resilient class 1 anti-RBD antibody, remains active against Omicron sub-variants up to BA.2.86. Cv2.3132, a near pan-Sarbecovirus neutralizer, targets the heptad repeat 2 membrane proximal region. When combined, Cv2.3194 and Cv2.3132 form a complementary SARS-CoV-2 neutralizing antibody cocktail exhibiting a local dose-dependent synergy. Thus, remarkably robust neutralizing memory B cell antibodies elicited in response to ancestral SARS-CoV-2 infection can withstand viral evolution and immune escape. The cooperative effect of such antibody combination may confer a certain level of protection against the latest SARS-CoV-2 variants., Competing Interests: The Institut Pasteur has pending patent applications on “Human neutralizing monoclonal antibodies against SARS-CoV-2 and their use thereof” (PCT/EP2022/058777, WO/2022/228827A1) in which C.P., I.F., T.B., X.M., F.A.R., O.S., and H.M. are inventors, and on “Combined antibodies against Sarbecoviruses and their use thereof” (EP23305528.4, PCT/IB2022/000108) in which C.P., T.B., O.S., and H.M. are inventors, both being licensed by the biotech company SpikImm. H.M. is a scientific consultant for SpikImm, and received consulting fees., (© 2024 The Authors.)

Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp -/f- ), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53 -/- mice are prone to tumor formation, we derived tumor cells from Trp53 -/- ;Tctp -/f- double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53 -/- ;Tctp -/f- mice show highly prolonged survival. Treatment of Trp53 -/- mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity., (© 2024. The Author(s).)

An antibody molecule that can bind to multiple distinct antigens is defined as polyreactive. In the present study, we performed statistical analyses to assess sequence correlates of polyreactivity of >600 antibodies cloned from different B-cell types of healthy humans. The data revealed several sequence patterns of variable regions of heavy and light immunoglobulin chains that determine polyreactivity. The most prominent identified patterns were increased number of basic amino acid residues, reduced frequency of acidic residues, increased number of aromatic and hydrophobic residues, and longer length of CDR L1. Importantly, our study revealed that antibodies isolated from different B-cell populations used distinct sequence patterns (or combinations of them) for polyreactive antigen binding. Furthermore, we combined the data from sequence analyses with molecular modeling of selected polyreactive antibodies and demonstrated that human antibodies can use multiple pathways for achieving antigen-binding promiscuity. These data reconcile some contradictions in the literature regarding the determinants of antibody polyreactivity. Moreover, our study demonstrates that the mechanism of polyreactivity of antibodies evolves during immune response and might be tailored to specific functional properties of different B-cell compartments. Finally, these data can be of use for efforts in the development and engineering of therapeutic antibodies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lecerf, Lacombe and Dimitrov.)

HIV-1 infection causes severe alterations of gut mucosa, microbiota and immune system, which can be curbed by early antiretroviral therapy. Here, we investigate how treatment timing affects intestinal memory B-cell and plasmablast repertoires of HIV-1-infected humans. We show that only class-switched memory B cells markedly differ between subjects treated during the acute and chronic phases of infection. Intestinal memory B-cell monoclonal antibodies show more prevalent polyreactive and commensal bacteria-reactive clones in late- compared to early-treated individuals. Mirroring this, serum IgA polyreactivity and commensal-reactivity are strongly increased in late-treated individuals and correlate with intestinal permeability and systemic inflammatory markers. Polyreactive blood IgA memory B cells, many of which egressed from the gut, are also substantially enriched in late-treated individuals. Our data establish gut and systemic B-cell polyreactivity to commensal bacteria as hallmarks of chronic HIV-1 infection and suggest that initiating treatment early may limit intestinal B-cell abnormalities compromising HIV-1 humoral response., (© 2023. Springer Nature Limited.)

Background: Emicizumab is a bispecific, chimeric, humanized immunoglobulin G (IgG)4 that mimics the procoagulant activity of factor (F) VIII (FVIII). Its long half-life and subcutaneous route of administration have been life-changing in treating patients with hemophilia A (HA) with or without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity; it prevents but does not treat acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors receiving emicizumab prophylaxis in whom exogenous FVIII is inefficient. We have shown recently that Imlifidase (IdeS), a bacterial IgG-degrading enzyme, efficiently eliminates human anti-FVIII IgG in a mouse model of severe HA with inhibitors and opens a therapeutic window for the administration of exogenous FVIII., Objectives: To investigate the impact of IdeS treatment in inhibitor-positive HA mice injected with emicizumab., Methods: IdeS was injected to HA mice reconstituted with human neutralizing anti-FVIII IgG and treated with emicizumab., Results: IdeS hydrolyzed emicizumab in vitro and in vivo, albeit, at slower rates than another recombinant human monoclonal IgG4. While F(ab') 2 fragments were rapidly cleared from the circulation, thus leading to a rapid loss of emicizumab procoagulant activity, low amounts of single-cleaved intermediate IgG persisted for several days. Moreover, the IdeS-mediated elimination of the neutralizing anti-FVIII IgG and restoration of the hemostatic efficacy of exogenous FVIII were not impaired by the presence of emicizumab and polyclonal human IgG in inhibitor-positive HA mice., Conclusion: Our results suggest that IdeS could be administered to inhibitor-positive patients with HA receiving emicizumab prophylaxis to improve and ease the management of breakthrough bleeds or programmed major surgeries., Competing Interests: Declaration of competing interests S.L.D. and J.D.D. are inventors on patent EP18305971.6 related to the use of IdeS in the context of AAV-mediated gene therapy. Other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Transplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn's immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules., Objectives: To investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta., Methods: We engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry., Results: Administration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery., Conclusion: Our results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life., Competing Interests: Declaration of competing interests J.M. and A.C.N. are inventors on a patent licensed to BioMarin. The remaining authors have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. All rights reserved.)

HIV-1 broadly neutralizing antibodies (bNAbs) can decrease viremia but are usually unable to counteract autologous viruses escaping the antibody pressure. Nonetheless, bNAbs may contribute to natural HIV-1 control in individuals off antiretroviral therapy (ART). Here, we describe a bNAb B cell lineage elicited in a post-treatment controller (PTC) that exhibits broad seroneutralization and show that a representative antibody from this lineage, EPTC112, targets a quaternary epitope in the glycan-V3 loop supersite of the HIV-1 envelope glycoprotein. The cryo-EM structure of EPTC112 complexed with soluble BG505 SOSIP.664 envelope trimers revealed interactions with N301- and N156-branched N-glycans and the 324 GDIR 327 V3 loop motif. Although the sole contemporaneous virus circulating in this PTC was resistant to EPTC112, it was potently neutralized by autologous plasma IgG antibodies. Our findings illuminate how cross-neutralizing antibodies can alter the HIV-1 infection course in PTCs and may control viremia off-ART, supporting their role in functional HIV-1 cure strategies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Independent of etiology, hemolytic diseases are associated with thrombosis, inflammation and immune dysregulation, all together contributing to organ damage and poor outcome. Beyond anemia and the loss of the anti-inflammatory functions of red blood cells, hemolysis leads to the release of damage-associated molecular patterns including ADP, hemoglobin, and heme, which act through multiple receptors and signaling pathways fostering a hyperinflammatory and hypercoagulable state. Extracellular free heme is promiscuous alarmin capable of triggering oxido-inflammatory and thrombotic events by inducing the activation of platelets, endothelial and innate cells as well as the coagulation and complement cascades. In this review, we discuss the main mechanisms by which hemolysis and, in particular, heme, drive this thrombo-inflammatory milieu and discuss the consequences of hemolysis on the host response to secondary infections., Competing Interests: Disclosures None.

Neutralizing anti-factor VIII (FVIII) antibodies, known as FVIII inhibitors, represent a major drawback of replacement therapy in persons with congenital hemophilia A (PwHA), rendering further infusions of FVIII ineffective. FVIII inhibitors can also appear in non-hemophilic individuals causing acquired hemophilia A (AHA). The use of non-FVIII bypassing agents in cases of bleeds or surgery in inhibitor-positive patients is complicated by the lack of reliable biological monitoring and increased thrombotic risk. Imlifidase (IdeS) is an endopeptidase that degrades human immunoglobulin G (IgG); it was recently approved for hyperimmune patients undergoing renal transplants. Here we investigated the ability of IdeS to eliminate FVIII inhibitors in vitro and in a model of inhibitor-positive HA mice. IdeS cleaved anti-FVIII plasma IgG from PwHA and AHA patients, and hydrolyzed recombinant human anti-FVIII IgG independently from their subclass or specificity for the A2, A3, C1 or C2 domains of FVIII. In HA mice passively immunized with recombinant human anti-FVIII IgG, IdeS restored the hemostatic efficacy of FVIII, as evidenced by the correction of the bleeding tendency. Our results provide the proof of concept for the transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy in inhibitor-positive patients.

SARS-CoV-2 infects cells by attachment to its receptor-the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation-, and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affects the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism and capacity to evade immune responses during evolution., (© 2022 The Protein Society.)

Heme regulates important biological processes by transient interactions with many human proteins. The goal of the present study was to assess extends of protein binding promiscuity of heme. To this end we evaluated interaction of heme with >9000 human proteins. Heme manifested high binding promiscuity by binding to most of the proteins in the array. Nevertheless, some proteins have outstanding heme binding capacity. Bioinformatics analyses revealed that apart from typical haemoproteins, these proteins are frequently involved in metal binding or have the potential to recognize DNA. This study can contribute for understanding the regulatory functions of labile heme., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

Broadly neutralizing antibodies against rapidly evolving viruses (e.g., HIV-1 and influenza virus), often manifest antigen-binding promiscuity. Based on a recent study, we hypothesize on the significance of antibody polyreactivity in neutralization of rapidly evolving viruses. We propose that polyreactivity contributes to toleration of viral variants and shortens the time for generating neutralizing antibodies., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The study of kinetics and thermodynamics of protein-protein interactions can contribute to assessment of the mechanism of molecular recognition process. These analyses can provide information about conformational changes and noncovalent forces that influence the initial recognition between proteins and stabilization of the complex. Studying these aspects may lead to a better comprehension of functions of proteins in biological environment and can become useful for the rational modification of some interactions by engineering of one of the implicated partners. Real-time biosensor assays based on surface plasmon resonance have been widely applied for the label-free evaluation of protein-protein interactions, allowing their characterization in term of binding affinity and kinetics. In the present chapter, we provide a protocol for the assessment of interactions involving complement proteins or antibodies, the protagonists of the immune system. We reported guidelines and indications concerning the analysis of the experimental data for the estimation of the kinetic parameters and for the evaluation of activation and equilibrium binding thermodynamics.

Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.

The typical functions of antibodies are based on linking the process of antigen recognition with initiation of innate immune reactions. With the introduction of modern research technologies and the use of sophisticated model systems, recent years have witnessed the discovery of a number of noncanonical functions of antibodies. These functions encompass either untypical strategies for neutralization of pathogens or exertion of activities that are characteristic for other proteins (cytokines, chaperones, or enzymes). Here, we provide an overview of the noncanonical functions of antibodies and discuss their mechanisms and implications in immune regulation and defense. A better comprehension of these functions will enrich our knowledge of the adaptive immune response and shall inspire the development of novel therapeutics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Natural antibodies (nAbs) are most commonly defined as immunoglobulins present in the absence of pathological conditions or deliberate immunizations. Occurrence of nAbs in germ- and antigen-free mice suggest that their production is driven, at least in part, by self-antigens. Accordingly, nAbs are constituted of natural autoantibodies (nAAbs), and can belong to the IgM, IgG, or IgA subclasses. These nAbs provide immediate protection against infection while the adaptive arm of the immune system mounts a specific and long-term response. Beyond immediate protection from infection, nAbs have been shown to play various functional roles in the immune system, which include clearance of apoptotic debris, suppression of autoimmune and inflammatory responses, regulation of B cell responses, selection of the B cell repertoires, and regulation of B cell development. These various functions of nAbs are afforded by their reactivity, which is broad, cross-reactive, and shown to recognize evolutionarily fixed epitopes shared between foreign and self-antigens. Furthermore, nAbs have unique characteristics that also contribute to their functional roles and set them apart from antigen-specific antibodies. In further support for the role of nAbs in the protection against infections and in the maintenance of immune homeostasis, the therapeutic preparation of polyclonal immunoglobulins, intravenous immunoglobulin (IVIG), rich in nAbs is commonly used in the replacement therapy of primary and secondary immunodeficiencies and in the immunotherapy of a large number of autoimmune and inflammatory diseases. Here, we review several topics on nAbs features and functions, and therapeutic applications in human diseases.

Background: Therapeutic normal IgG or intravenous immunoglobulin (IVIG) exerts anti-inflammatory effects through several mutually nonexclusive mechanisms. Recent data in mouse models of autoimmune disease suggest that IVIG induces IL-4 in basophils by enhancing IL-33 in SIGN-related 1-positive innate cells. However, translational insight on these data is lacking., Objective: We sought to investigate the effect of IVIG on human basophil functions., Methods: Isolated circulating basophils from healthy donors were cultured in the presence of IL-3, IL-33, GM-CSF, thymic stromal lymphopoietin, or IL-25. The effect of IVIG and F(ab') 2 and Fc IVIG fragments was examined based on expression of various surface molecules, phosphorylation of spleen tyrosine kinase, induction of cytokines, and histamine release. Basophil phenotypes were also analyzed from IVIG-treated patients with myopathy. Approaches, such as depletion of anti-IgE reactivity from IVIG, blocking antibodies, or inhibitors, were used to investigate the mechanisms., Results: We report that IVIG directly induces activation of IL-3-primed human basophils, but IL-33 and other cytokines were dispensable for this effect. Activation of basophils by IVIG led to enhanced expression of CD69 and secretion of IL-4, IL-6, and IL-8. IVIG-treated patients with myopathy displayed enhanced expression of CD69 on basophils. The spleen tyrosine kinase pathway is implicated in these functions of IVIG and were mediated by F(ab') 2 fragments. Mechanistically, IVIG induced IL-4 in human basophils by interacting with basophil surface-bound IgE but independent of FcγRII, type II Fc receptors, C-type lectin receptors, and sialic acid-binding immunoglobulin-like lectins., Conclusion: These results uncovered a pathway of promoting the T H 2 response by IVIG through direct interaction of IgG with human basophils., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Antibodies are essential components of adaptive immunity. A typical antibody repertoire comprises an enormous diversity of antigen-binding specificities, which are generated by the genetic processes of recombination and mutation. Accumulating evidence suggests that the immune system can exploit additional strategies to diversify the repertoire of antigen specificities. These unconventional mechanisms exclusively target the antigen-binding sites of immunoglobulins and include the insertion of large amino acid sequences, post-translational modifications, conformational heterogeneity and use of nonprotein cofactor molecules. Here, we describe the different unconventional routes for diversification of antibody specificities. Furthermore, we highlight how the immune system has a much greater level of adaptability and plasticity than previously anticipated, which goes far beyond that encoded in the genome or generated by the acquisition of somatic mutations.

Objectives: We evaluated the potential clinical relevance of malondialdehyde (MDA) and autoantibodies to copper oxidized low-density lipoprotein (CuOx-LDL) in type 2 diabetes occurrence., Methods: This cross-sectional study enrolled 69 normoglycemic subjects, 18 prediabetic patients and 108 type 2 diabetes patients. MDA concentration was assessed spectrophotometrically. Plasma IgG, IgA and IgM levels to CuOx-LDL were determined by ELISA., Results: Plasma MDA levels were considerably higher in obese, prediabetic and type 2 diabetes subjects compared to controls. In multiple linear regression analysis, both MDA and IgA to CuOx-LDL were significantly associated with glucose metabolism markers (p<0.05). Multiple logistic regression analyses showed that high plasma MDA and IgA to CuOx-LDL were independent risk factors for type 2 diabetes (OR 1.196, 95% CI: 1.058 to 1.353; p=0.004; OR 1.626, 95% CI: 1.066 to 2.481; p=0.024; respectively). Importantly, elevated IgA to CuOx-LDL predicted incident diabetes in patients with prediabetes (OR 2.321, 95% CI:1.063 to 5.066; p=0.035). From stratified analyses by body mass index (BMI), both MDA and IgA to CuOx-LDL remained independent predictors of type 2 diabetes occurrence in non-obese subjects (p<0.05). More interesting, elevated IgA to CuOx-LDL levels could be predictors of type 2 diabetes in obese prediabetic subjects (p=0.044). Conversely, neither IgG nor IgM to CuOx-LDL was associated with glucose metabolism markers, obesity or type 2 diabetes., Conclusions: Plasma MDA and IgA to CuOx-LDL were significantly associated with blood markers of glucose metabolism. High levels of MDA and IgA to CuOx-LDL could independently predict type 2 diabetes development in normoglycemia and prediabetic subjects., (Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.)

Antibody repertoires of healthy humans and animals contain a fraction of antibodies able to acquire additional polyspecificity following exposure to several biologically relevant redox molecules (free heme, reactive oxygen species, ferrous ions, HOCl, etc.). The physiological role of these "hidden" polyspecific antibodies is poorly understood. Similar to inherently polyspecific antibodies, those with induced polyspecificicty may also have immunoregulatory properties. We have previously shown that a pooled human IgG preparation, modified by the exposure to ferrous ions, acquires the ability to significantly improve survival of animals with polymicrobial sepsis or aseptic systemic inflammation induced by bacterial lipopolysaccharide or zymosan administration. In the present study, we have analyzed the effects of administration of heme-exposed pooled human IgG in the same models of sepsis and aseptic systemic inflammation. The administration of a single dose of heme-exposed pooled IgG has resulted in a significant increase in the survival of mice with endotoxinemia, but not in those with polymicrobial sepsis and zymosan-induced severe generalized inflammation. Finally, we have provided evidence that the anti-inflammatory effect of heme-exposed IgG can be explained by scavenging of pro-inflammatory mediators.

Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)n polymorphism and single nucleotide polymorphisms located at -1135 and -413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46-3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30-3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development.

Polyspecificity (polyreactivity) is currently considered an intrinsic property of a subset of antibodies, primarily of naturally occurring autoantibodies. Polyspecificity is no longer viewed as a biologically irrelevant stickiness. Furthermore, the capacity to bind defined sets of unrelated antigens finds its structural explanation. What is most intriguing, the elucidation of the role of polyspecificity may promote a better understanding of specific recognition as a function of the entire immune system. The early events of immune recognition depend on polyspecific binding. Thus, the completeness of the naïve repertoires of antigen receptors is ensured. The process of immunologically-relevant antigen recognition that is initiated goes beyond simple molecular interaction with the antigenic determinants. It involves cellular cooperation and culminates in antibody response maturation. Recent findings also pave the way for the clinical application of posttranslationally induced polyspecificity.

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.