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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 affect 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.

International audience; Therapeutic antibodies should cover particular physicochemical and functional requirements for successful entry into clinical practice. Numerous experimental and computational approaches have been developed for early identification of different unfavourable features of antibodies. Immune repertoires of healthy humans contain a fraction of antibodies that recognize nitroarenes. These antibodies have been demonstrated to manifest antigen-binding polyreactivity. Here we observed that >20 % of 112 clinical stage therapeutic antibodies show pronounced binding to 2,4-dinitrophenol conjugated to albumin. This interaction predicts a number of unfavourable functional and physicochemical features of antibodies such as polyreactivity, tendency for self-association, stability and expression yields. Based on these findings we proposed a simple approach that may add to the armamentarium of assays for early identification of developability liabilities of antibodies intended for therapeutic use.

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 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.

International audience; Therapeutic antibodies have transformed the clinical practice. Not surprisingly, development of antibody thera-peutics is currently the main focus of the biotechnology industry. Nonetheless, the development process is complex , and many antibodies do not reach the clinic. Reasons for the failures include, undesired binding behavior (polyreactivity), low stability, poor expression yields, unfavorable pharmacokinetics etc. Numerous studies have proposed different analytical methods for assessment of physicochemical parameters of antibodies and identification of problematic molecules at early stages of the development process. These studies, however, have not addressed the basic mechanistic question of how sequence features of variable regions determinate the different biophysical characteristics and the binding behavior of the antibodies. In a recent study, Jain et al assessed 12 biophysical qualities of 137 monoclonal therapeutic antibodies. We used the raw data from this comprehensive study to perform correlation analyses of different biophysical measurables with various sequence features of variable regions of the antibodies-number of mutations, length of hypervariable loops, and frequency of amino acid residue types. The obtained data reveled significant relationships between the sequence characteristics of the variable domains and different physiochemical properties of antibodies. The data from this study can assist in design of a set of criteria for early identification of antibodies with developability issues. Moreover, our findings provide novel fundamental insights into the sequence-function relationship of antibodies.

Immunoglobulin repertoires contain a fraction of antibodies that recognize low molecular weight compounds, including some enzymes’ cofactors, such as heme. Here, by using a set of 113 samples with variable region sequences matching clinical-stage antibodies, we demonstrated that a considerable number of these antibodies interact with heme. Antibodies that interact with heme possess specific sequence traits of their antigen-binding regions. Moreover they manifest particular physicochemical and functional qualities i.e. increased hydrophobicity, higher propensity of self-binding, higher intrinsic polyreactivity and reduced expression yields. Thus, interaction with heme is a strong predictor of different molecular and functional qualities of antibodies. Notably, these qualities are of high importance for therapeutic antibodies, as their presence was associated with failure of drug candidates to reach clinic. Our study reveled an important facet of information about relationship sequence-function in antibodies. It also offers a convenient tool for detection of liabilities of therapeutic antibodies., Lecerf et al. examine over 100 samples with variable region sequences matching clinical-stage immunoglobulin antibodies, to demonstrate that many interact with heme. They find that those that do interact with heme possess specific sequence traits that manifest qualities such as increased hydrophobicity, self-binding and intrinsic polyreactivity, thus suggesting that heme interaction is a predictor for therapeutically important qualities.

Neonatal Fc receptor (FcRn) has a key role in the homeostasis of IgG. Despite its physiological and clinical importance, the interaction of IgG and FcRn remains not completely comprehended. Thus, IgG molecules with identical constant portions but with minor differences in their V regions have been demonstrated to interact with FcRn with a considerable heterogeneity in the binding affinity. To understand this discrepancy, we dissected the physicochemical mechanism of the interaction of 10 human IgG1 to human FcRn. The interactions of two Abs in the presence of their cognate Ags were also examined. Data from activation and equilibrium thermodynamics analyses as well as pH dependence of the kinetics revealed that the V region of IgG could modulate a degree of conformational changes and binding energy of noncovalent contacts at the FcRn binding interface. These results suggest that the V domains modulate FcRn binding site in Fc by allosteric effects. These findings contribute for a deeper understanding of the mechanism of IgG–FcRn interaction. They might also be of relevance for rational engineering of Abs for optimizing their pharmacokinetic properties.

Autophagy plays an important role in the regulation of autoimmune and autoinflammatory responses of the immune cells. Defective autophagy process is associated with various autoimmune and inflammatory diseases. Moreover, in many of these diseases, the therapeutic use of normal immunoglobulin G or intravenous immunoglobulin (IVIG), a pooled normal IgG preparation, is well documented. Therefore, we explored if IVIG immunotherapy exerts therapeutic benefits via induction of autophagy in the immune cells. Here we show that IVIG induces autophagy in peripheral blood mononuclear cells (PBMCs). Further dissection of this process revealed that IVIG-induced autophagy is restricted to inflammatory cells like monocytes, dendritic cells, and M1 macrophages but not in cells associated with Th2 immune response like M2 macrophages. IVIG induces autophagy by activating AMP-dependent protein kinase, beclin-1, class III phosphoinositide 3-kinase and p38 mitogen-activated protein kinase and by inhibiting mammalian target of rapamycin. Mechanistically, IVIG-induced autophagy is F(ab′)2-dependent but sialylation independent, and requires endocytosis of IgG by innate cells. Inhibition of autophagy compromised the ability of IVIG to suppress the inflammatory cytokines in innate immune cells. Moreover, IVIG therapy in inflammatory myopathies such as dermatomyositis, antisynthetase syndrome and immune-mediated necrotizing myopathy induced autophagy in PBMCs and reduced inflammatory cytokines in the circulation, thus validating the translational importance of these results. Our data provide insight on how circulating normal immunoglobulins maintain immune homeostasis and explain in part the mechanism by which IVIG therapy benefits patients with autoimmune and inflammatory diseases.

International audience; In humans, maternal IgGs are transferred to the fetus from the second trimester of pregnancy onwards. The transplacental delivery of maternal IgG is mediated by its binding to the neonatal Fc receptor (FcRn) after endocytosis by the syncytiotrophoblast. IgGs present in the maternal milk are also transferred to the newborn through the digestive epithelium upon binding to the FcRn. Importantly, the binding of IgGs to the FcRn is also responsible for the recycling of circulating IgGs that confers them with a long half-life. Maternally delivered IgG provides passive immunity to the newborn, for instance by conferring protective anti-flu or anti-pertussis toxin IgGs. It may, however, lead to the development of autoimmune manifestations when pathological autoantibodies from the mother cross the placenta and reach the circulation of the fetus. In recent years, strategies that exploit the transplacental delivery of antigen/IgG complexes or of Fc-fused proteins have been validated in mouse models of human diseases to impose antigen-specific tolerance, particularly in the case of Fc-fused factor VIII (FVIII) domains in hemophilia A mice or pre-pro-insulin (PPI) in the case of preclinical models of type 1 diabetes (T1D). The present review summarizes the mechanisms underlying the FcRn-mediated transcytosis of IgGs, the physiopathological relevance of this phenomenon, and the repercussion for drug delivery and shaping of the immune system during its ontogeny.

The standard assay for characterization of interaction of heme with proteins is absorbance spectroscopy. However, this approach demands relatively large quantities of proteins and it is difficult to perform in high-throughput manner. Here, we describe an immunosorbent assay based on the covalent in situ conjugation of heme to a pre-coated carrier. Advantage of this assay is that it allows both identification of heme-binding proteins and quantification of their binding avidity, using only minimal amounts of protein (1-10 μg). Importantly, the same approach can be used for covalent linkage of other natural or synthetic compounds and analyzing their interactions with proteins.

In a healthy immune repertoire, there exists a fraction of polyreactive antibodies that can bind to a variety of unrelated self- and foreign antigens. Apart from naturally polyreactive antibodies, in every healthy individual, there is a fraction of antibody that can gain polyreactivity upon exposure to porphyrin cofactor heme. Molecular mechanisms and biological significance of the appearance of cryptic polyreactivity are not well understood. It is believed that heme acts as an interfacial cofactor between the antibody and the newly recognized antigens. To further test this claim and gain insight into the types of interactions involved in heme binding, we herein investigated the influence of a group of aromatic guanylhydrazone molecules on the heme-induced antibody polyreactivity. From the analysis of SAR and the results of UV-vis absorbance spectroscopy, it was concluded that the most probable mechanism by which the studied molecules inhibit heme-mediated polyreactivity of the antibody is the direct binding to heme, thus preventing heme from binding to antibody and/or antigen. The inhibitory capacity of the most potent compounds was substantially higher than that of chloroquine, a well-known heme binder. Some of the guanylhydrazone molecules were able to induce polyreactivity of the studied antibody themselves, possibly by a mechanism similar to heme. Results described here point to the conclusion that heme indeed must bind to an antibody to induce its polyreactivity, and that both π-stacking interactions and iron coordination contribute to the binding affinity, while certain structures, such as guanylhydrazones, can interfere with these processes. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3794]

International audience; Therapeutic intravenous immunoglobulin preparations (IVIg) are used for treatment of wide range of auto-immune and inflammatory diseases. Versatile mechanisms have been reported to contribute to the im-munomodulatory effects of IVIg. Here we demonstrate that IVIg has a strong potential to inhibit pro-in-flammatory effect of extracellular heme. Indeed, the presence of immunoglobulins reduced the potential of heme to activate the complement system on the surface of human endothelial cells. Since extracellular heme is considered as one of the principal pathogenic factors in hemolytic disorders, its therapeutic scavenging by IVIg may have significant clinical repercussions.

International audience; Objective: Antibodies with catalytic (hydrolytic) properties to DNA or RNA have been reported in systemic lupus erythematosus (SLE). However, it is well known that ethnicity plays an important role in the presentation of SLE and severity of the disease; hence, these data may not truly represent a general feature of all SLE patients. Therefore, we have analyzed the hydrolyzing activity of immunoglobulin G (IgG) of SLE patients from the Indian population with an aim to decode whether the catalytic antibody response represents part of an active disease process.Methods: IgGs were isolated from the sera of 72 consecutive patients diagnosed with SLE. As a control, IgGs from healthy donors were used. The catalytic activity of IgG was measured by PFR-MCA and affinity-linked oligonucleotide nuclease assay.Results: IgGs from patients with SLE from the Indian subcontinent displayed significantly higher hydrolysis rates of both the surrogate substrate, PFR-MCA, and the DNA than IgG from healthy individuals. Intergroup comparisons of the IgG-PFR-MCA interactions with clinical manifestations of the disease demonstrated a significantly increased level of hydrolysis among the patients with renal involvement who tested positive for anti-dsDNA antibodies. The PFR-MCA hydrolysis also appears to be associated with the active disease (p=0.0988, vs. inactive group).Conclusion: The prevalence of catalytic antibodies represents a general feature of SLE patients, irrespective of their origin.

The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses.

An antibody molecule that recognizes multiple unrelated antigens is defined as polyreactive. Polyreactivity is an intrinsic characteristic of immune repertoires. Degenerated antigen binding diversifies the repertoire of specificities, thus contributing to immune defense and immune regulation. Immune repertoire contains also a fraction of immunoglobulins, which acquire polyreactivity only following contact with various protein-destabilizing or pro-oxidative substances. Posttranslational induction of the antibody polyreactivity may have important repercussion for laboratory practice, as well as in cases of pathological conditions accompanied by liberation of large quantities of pro-oxidative substances such as heme, labile iron, or reactive oxygen species. Antibodies with induced polyreactivity have been demonstrated to exert pathogen neutralization and immune regulatory potential in inflammatory conditions, suggesting that this phenomenon may be exploited for design of therapeutic strategies. In this article, we provide description of the basic procedures for uncovering of the cryptic polyreactivity of antibodies by heme, ferrous ions, and acid pH solution.

An antibody molecule that recognizes multiple unrelated antigens is defined as polyreactive. Polyreactivity is an intrinsic characteristic of immune repertoires. Degenerated antigen binding diversifies the repertoire of specificities, thus contributing to immune defense and immune regulation. Immune repertoire contains also a fraction of immunoglobulins, which acquire polyreactivity only following contact with various protein-destabilizing or pro-oxidative substances. Posttranslational induction of the antibody polyreactivity may have important repercussion for laboratory practice, as well as in cases of pathological conditions accompanied by liberation of large quantities of pro-oxidative substances such as heme, labile iron, or reactive oxygen species. Antibodies with induced polyreactivity have been demonstrated to exert pathogen neutralization and immune regulatory potential in inflammatory conditions, suggesting that this phenomenon may be exploited for design of therapeutic strategies. In this article, we provide description of the basic procedures for uncovering of the cryptic polyreactivity of antibodies by heme, ferrous ions, and acid pH solution.

Polyreactive antibodies represent a significant fraction of immune repertoires and play an important role in the immune defense and immune homeostasis. Polyreactive B-cell receptors (BCR), however, are frequently expressed by B-cell lymphomas. It was suggested that polyreactive BCR on lymphoma cells might deliver stimulation signals by binding to various endogenous or exogenous antigens, thus promoting the survival of the malignant cells. In addition to natural polyreactive antibodies, immune repertoires contain antibodies that acquire polyreactivity after exposure to different redox-active substances such as reactive oxygen species, iron ions and heme. Here, we demonstrate that an antibody cloned from a patient's splenic marginal zone B-cell lymphoma acquires physiologically relevant binding affinity to various autoantigens following exposure to heme. We elucidated the mechanisms underlying polyreactive antigen binding. The results obtained in this study imply that antigen-binding receptors expressed on some malignant cells acquire polyreactivity after exposure to redox substances that are released at sites of inflammation or as a result of cellular damage. The acquisition of novel BCR specificities under hemolytic or inflammatory conditions may play an important role in the physiopathology of certain B-cell malignancies.

Intravenous immunoglobulin (IVIg) is a pooled preparation of polyspecific, polyreactive immunoglobulin (Ig)G molecules from several thousand healthy donors. The immunoglobulin (Ig) molecule has Fab region, which is involved in antigen-binding and the Fc portion, which is involved in effector function. As IVIg is prepared from multiple donors, it contains numerous antibodies directed against a wide range of antigens; consequently, the variable regions on the Ig Fab fragments in IVIg preparations are diverse. The variable region can bind to non-self-antigens (foreign antigens), self-antigens and anti-idiotypic antibodies. IVIg contains a broad spectrum of antibody specificities against bacterial, viral, parasitic and mycoplasma antigens, that are capable of both opsonization and neutralization of microbes and toxins. In addition to its initial use as replacement therapy in primary and secondary immunodeficiencies, IVIg is widely indicated in a large spectrum of autoimmune and inflammatory diseases. One of the first proposed mechanisms of action of IVIg was via Fcγ receptor blockade 1. This study demonstrated that infusion of Fcγ fragments in idiopathic thrombocytopenic purpura/immune thrombocytopenia (ITP) patients increased platelet count, mediated by the blockade of Fcγ receptors 1. It has since been demonstrated in vivo that Fcγ fragments, particularly if sialylated, can exert anti-inflammatory effects 2. This suggests that the clinical benefits of IVIg may be mediated via an Fc pathway; indeed, to date there have been no studies that demonstrate the clinical benefit of Fab fragments alone. However, non-Fc mechanisms have been proposed that provide an insight into the possible molecular mechanisms of action of IVIg, although these do not exclude the potential co-operation of Fab and Fc portion of IgG to elicit the effects of IVIg. One mechanism of action was proposed by Sultan et al., who found that anti-idiotypic antibodies in IVIg were effective in the treatment of autoimmune haemophilia 3. This led to the study and characterization of anti-idiotypic antibodies in IVIg which neutralize pathogenic autoantibodies 4,5. IVIg was found to contain anti-idiotypes against anti-factor VIII, anti-neutrophil cytoplasmic antibody, anti-DNA, anti-thyroglobulin, anti-acetylcholine receptors and anti-neuroblastoma antigens. Furthermore, anti-idiotypic antibodies have been found to play a role in transplantation due to the anti-human leucocyte antigen antibodies. In addition to ITP and haemophilia, IVIg has been found to be effective in several inflammatory and autoimmune diseases. Therefore, anti-inflammatory effects of IVIg were studied and were shown to be mediated in part through anti-complement effects. Dermatomyosis is a condition mediated by C5b/C9 membranolytic attack complexes (MACs) in intramuscular capillaries. The formation of MAC occurs when C3 is hydrolyzed into C3b, which leads to the activation of C5b and the formation of MACs. In a study conducted by Basta et al. 6, IVIg was found to form complexes with C3, preventing MAC formation and deposition in patients with dermatomyosis. This suggests that the clinical benefit of IVIg can be attributed to complement scavenging, demonstrating an additional distinct mechanism of action that may be mediated by F(ab′)2 and whole IVIg, but not Fcγ fragments alone. Anaphylatoxins are complement peptides that are produced when the complement system is activated. A study by Basta et al. implicates F(ab′)2 in the neutralization of anaphylatoxins, such as C3a and C5a 7. IVIg is able to suppress C3a- and C5a-induced release of thromboxane B2 and histamine, which have proinflammatory properties. Moreover, circulatory collapse caused by C5a was prevented in pigs pretreated with F(ab′)2 IVIg. The neutralization of C3a and C5a were observed in cells treated with F(ab′)2 IVIg and whole IVIg and not Fcγ IVIg fragments, suggesting that F(ab′)2 and not Fcγ are implicated in this process. IVIg has also been found to be beneficial in a murine model of brain ischaemia and stroke, via a complement scavenging mechanism. Administration of IVIg, either prior to an ischaemic event or during reperfusion, led to a two- to three-fold improvement in functional outcomes in ischaemia and reperfusion. C3 levels were higher in injured compared to non-injured brain regions. Furthermore, compared with wild-type mice, C5-deficient mice were protected from ischaemia and reperfusion. IVIg decreased C3 and caspase 3 activation, suggesting that IVIg inhibits complement-mediated cell damage via scavenging of complement proteins to elicit beneficial effects 8. In addition to a role in scavenging complement in inflammatory and immune diseases, IVIg has also been shown to alter the cytokine network and mediate the balance between T helper (Th) types. Th cells can be classified into several subsets, such as Th1, Th2, Th17 and regulatory T cells, which produce distinct cytokines. Th1 cells produce cytokines such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α, Th2 cells produce IL-4, IL-5, IL-13 and IL-10, Th17 cells produce IL-17, IL-21 and IL-22, and regulatory T cells which are immunosuppressor cells produce TGF-β and IL-10. In a study conducted by Ruiz de Souza et al., peripheral blood monocytes treated with IVIg induced an up-regulation of anti-inflammatory cytokine IL-1 receptor antagonist and down-regulation of several proinflammatory cytokines 9. By the early 2000s there was an increasing focus on the role of dendritic cells and their effect on T cell polarization. Mature dendritic cells can stimulate naive T helper cells (Th0) and polarize them into distinct subsets. Our study demonstrated that both the F(ab′)2 and Fc fragments of IVIg are capable of inhibiting the differentiation and maturation of dendritic cells, suggesting that IVIg is capable of inducing tolerogenic dendritic cell phenotypes 10. As a consequence of IVIg-induced tolerogenic dendritic cells, regulatory T cells are up-regulated. Using a murine model of autoimmune encephalomyelitis (EAE), prophylactic IVIg was found to increase CD4+CD25+forkhead box protein 3 (FoxP3+) regulatory T cells 11. This proliferation of regulatory T cells has also been observed in humans following high-dose IVIg treatment in patients with autoimmune rheumatic disease 12. We recently reported that, in EAE mice, IVIg inhibits the differentiation of CD4+ T cells to Th1 and Th17 cells 13. The down-regulation of Th1 and Th17 cells was observed with a concomitant up-regulation of regulatory T cells, demonstrating the reciprocal regulation mechanism of IVIg. Furthermore, the reciprocal regulation was suggested to be F(ab′)2-dependent due to the comparable inhibition of Th1 and Th17 cells observed in mice treated with F(ab′)2 fragments or IVIg 13. IVIg-induced expansion of regulatory T cells may be due to several mechanisms. Mazer et al. propose that IVIg renders dendritic cells tolerogenic via its interaction with dendritic cell immunoreceptor (DCIR) 14. This leads to increased levels of FoxP3+ regulatory T cells which can attenuate autoimmune disease severity. Another mechanism of action for regulatory T cell expansion is provided recently by our group. Our report suggests that IVIg-induced expansion of regulatory T cells is due to the induction of cyclo-oxygenase 2-dependent prostaglandin E2 production in dendritic cells 15. Inhibition of cyclo-oxygenase 2 enzymatic activity significantly reduced IVIg-mediated regulatory T cell expansion both in vitro and in vivo in EAE mice. This mechanism was dependent on Fab fragments of IVIg but not Fc. Immunomodulatory mechanisms of IVIg in autoimmune conditions are not fully understood, although several mutually non-exclusive effects have been proposed. Individually, each of these mechanisms may participate to a certain extent in the overall effect of IVIg. While some of the effects may rely upon the binding of the Fc moiety of IgG to Fcγ receptors on target cells, others may be primarily dependent on the range of variable regions of IgG.

International audience; The programed death-1 (PD-1)–programed death ligand-1 (PD-L1) and PD-L2 co-inhib-itory pathway has been implicated in the evasion strategies of Mycobacterium tuberculosis. Specifically, M. tuberculosis-induced PD-L1 orchestrates expansion of regulatory T cells and suppression of Th1 response. However, the role of PD pathway in regulating Th17 response to M. tuberculosis has not been investigated. In the present report, we demonstrate that M. tuberculosis and M. tuberculosis-derived antigen fractions have differential abilities to mediate human monocyte-and dendritic cell (DC)-mediated Th17 response and were independent of expression of PD-L1 or PD-L2 on aforementioned antigen-presenting cells. Importantly, we observed that blockade of PD-L1 or PD-1 did not significantly modify either the frequencies of Th17 cells or the production of IL-17 from CD4 + T cells though IFN-γ response was significantly enhanced. On the contrary, IL-1β from monocytes and DCs were critical for the Th17 response to M. tuberculosis. Together, our results indicate that IL-1β, but not members of the programed death pathway , is critical for human Th17 response to M. tuberculosis.

To identify the hematological manifestations and its association with serum ferritin levels in SLE patients from Western India.Ninety clinically diagnosed SLE patients fulfilling ACR criteria were included. Disease activity was assessed at the time of evaluation using Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Sera were tested for serum ferritin levels by ELISA (Calbiotech, USA). Autoantibodies such as ANA, anti-dsDNA by indirect immunofluorescence test (IFA- Bio-Rad, USA) and anti-cardiolipin antibodies (ACA) to IgG and IgM isotypes and Anti-β2 GP antibodies to IgG and IgM isotypes were detected by ELISA using commercially available kits (Euroimmun, Lubeck, Germany).Out of 90 SLE patients studied, 41 patients (45.6%) showed hematological abnormalities, where anemia (82.9%), leucopenia (26.8%), autoimmune hemolytic anemia (AIHA) (14.6%) and idiopathic thrombocytopenic purpura (ITP) were noted in (34.1%) patients. Mean±SD serum ferritin levels among SLE patients were 270.2±266.0 ng/ml as compared to 29.0±15.8 ng/ml healthy normal controls (p0.0001). A positive correlation between serum ferritin levels and SLEDAI scores (r= 0.2640, p=0.0124) and anti-dsDNA positivity was noted (r=0.32, p0.0001). Serum ferritin levels were negatively correlated with hemoglobin levels (r=-0.5964, p=0.0001), WBC count (r=-0.1705, p=0.2316), platelet count ((r=-0.1701, P=0.2375), C3 levels (r=-0.4417, p=0.0034) and C4 levels (r=-0.0363, p=0.8215).Serum ferritin is an excellent marker of SLE which can be used for an evaluation of disease activity particularly in active stage of the disease mainly in patients having hematological and renal manifestations.

Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP-MurNAc-pentapeptide, respectively. Synthesis of 2'-azido RNA helix starts from 2'-azido-2'-deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22-nt RNA helix mimicking the acceptor arm of Ala-tRNA(Ala) by T4 RNA ligase. For alkyne UDP-MurNAc-pentapeptide, meso-cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L-Cys is converted to dehydroalanine with O-(mesitylenesulfonyl)hydroxylamine. Reaction of but-3-yne-1-thiol with dehydroalanine affords the alkyne-containing UDP-MurNAc-pentapeptide. The Cu(I)-catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine provided the peptidyl-RNA conjugate, which was tested as an inhibitor of non-ribosomal FemX(Wv) aminoacyl transferase. The bi-substrate analogue was found to inhibit FemX(Wv) with an IC(50) of (89±9) pM, as both moieties of the peptidyl-RNA conjugate contribute to high-affinity binding.

Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemX(Wv). Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemX(Wv) with the acceptor arm of tRNA(Gly) and with L-Ser or larger residues quantitatively accounts for the preferential transfer of L-Ala observed with complete aminoacyl-tRNAs. The main FemX(Wv) identity determinant was identified as the penultimate base pair (G(2)-C(71)) of the acceptor arm instead of G(3)*U(70) for the alanyl-tRNA synthetase. FemX(Wv) tolerated a configuration inversion of the Calpha of L-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemX(Wv) is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNA(Gly) from the active site.

Eight Y chromosome short tandem repeats (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385I/II) were used to assess haplotype distribution in non-selected, unrelated Bantu males living in Central Africa [N. Mathias, M. Bayes, C. Tyler-Smith, Highly informative compound haplotypes for the human Y chromosome, Hum. Mol. Genet. 3 (1994) 115-123; L. Roewer, J. Arnemann, N.K. Spurr, K.H. Grzeschik, J.T. Epplen, Simple repeat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts, Hum. Genet. 89 (1992) 389-394; P. De Knijff, M. Kayser, A. Caglia, D. Corach, N. Fretwel, C. Gehrig, G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M. Hidding, K. Honda, M. Jobling, M. Krawczak, K. Leim, S. Meuser, E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G. Penacino, A. Perez-Lezaun, A. Piccini, M. Prinz, C. Schmitt, P. M. Schneider, R. Szibor, J. Teifel-Greding, G. Weishold, L. Rower, Chromosome Y microsatellites: population genetic and evolutionary aspects, Int. J. Legal Med. 110 (1997) 134-149; M. Kayser, A. Caglia, D. Corach, N. Fretwel, C. Gehrig, G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M. Hidding, K. Honda, M. Jobling, M. Krawczak, K. Leim, S. Meuser, E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G. Penacino, A. Perez-Lezaun, A. Piccini, M. Prinz, C. Schmitt, P. M. Schneider, R. Szibor, J. Teifel-Greding, G. Weishold, P. de Knijff, L. Rower, Evaluation of Y chromosome STRs: a multicenter study, Int. J. Legal Med. 110 (1997) 125-133, 141-149]. One hundred and sixty-five full haplotypes were obtained from Bantu males. The most common haplotype (DYS19-15, DYS389I-13, DYS389II-30, DYS390-21, DYS391-10, DYS392-11, DYS393-13, DYS385I/II-15,17) was shared by 5.5% of individuals. In the Bantu population in Central Africa, the haplotype diversity and the discrimination capacity of Y-STR may be estimated at 99.14% and 0.5333, respectively.

International audience; Central tolerance plays a key role in modulating immune responses to self and exogenous antigens. The absence of self-antigen expression, as in patients with genetic deficiencies, prevents the development of antigen-specific immune tolerance. Hence, a substantial number of patients develop neutralizing antibodies to the corresponding protein therapeutics after replacement treatment. In this context, the administration of missing antigens during fetal development, a key period for self-tolerance establishment, should confer early and long-lasting antigen-specific tolerance. To this end, we exploited the physiological pathway of the neonatal Fc receptor (FcRn) through which maternal immunoglobulins are transplacentally transferred to fetuses. We demonstrate that Fc-fused antigens administered to pregnant mice reach fetal lymphoid organs in an FcRn-dependent manner, accumulate in antigen-presenting cells of myeloid origin, and promote the generation of both thymic and peripheral antigen-specific regulatory T cells. This strategy was successfully pursued in a mouse model of hemophilia A, where maternofetal transfer of the Fc-fused immunodominant domains of coagulation factor VIII conferred antigen-specific tolerance. Transplacental tolerance induction with Fc-fused proteins may thus prove valuable to prevent alloimmunization after replacement protein therapy for congenital deficiencies.

Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis.

Background. Systemic sclerosis (SSc, scleroderma) is a disorder characterized by fibrosis of skin and visceral organs. Pathogenesis of scleroderma is complex and is incompletely understood as yet. Autoantibodies in SSc represent a serologic hallmark which have clinical relevance, with diagnostic and prognostic potential.Objectives. To study distribution of clinical manifestations and to identify frequency of autoantibodies among subtypes of scleroderma patients from Western India.Methodology. One hundred and ten scleroderma patients were clinically classified according to the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria. All these patients were in active stage of disease. Clinical manifestations were recorded at the time of presentation. Autoantibodies were tested in them by indirect immunofluorescence test and ELISA. Immunoglobulin levels were estimated by nephelometer. These parameters were further correlated with clinical presentation of the disease.Results. Scleroderma patients had M : F ratio of 1 : 10 where mean age at evaluation was34.7±10.7years and a mean disease duration was43.7±35months. Clinical subtypes showed that 45 patients (40.9%) had diffused cutaneous (dcSSc) lesions, 32 patients (29.1%) had limited cutaneous (lcSSc) lesions, and 33 patients (30%) had other autoimmune overlaps. The overall frequency of ANA in SSc patients studied was 85.5%. The frequency of anti-Scl70, anti-centromere, anti-endothelial cell antibodies (AECA), and anti-keratinocyte antibodies (AKA) was 62.7%, 22.7%, 30%, and 40.9%, respectively. Anti-Scl70 antibodies were significantly high (75.6% versus 46.9%) among dcSSc patients (P<0.0115) whereas anti-centromere antibodies were significantly high (9% versus 38%) among lcSSc patients when these two subtypes were compared (P<0.0044).Conclusion. This study supports that there are geoepidemiological variations among scleroderma patients for their clinical presentation, autoantibody profile, and immune parameters across the country.

The mechanisms underlying Japanese encephalitis virus (JEV) pathogenesis need to be thoroughly explored to delineate therapeutic approaches. It is believed that JEV manipulates the innate and adaptive compartments of the host's immune system to evade immune response and cross the blood-brain barrier. The present study was thus designed to investigate the functional modulation of DCs after exposure to JEV and to assess the consequences on CD4(+) T-lymphocyte functions. Human monocyte-derived DCs were either infected with 1 MOI of live virus, UV-inactivated virus, or were mock-infected. Replication-competent JEV induced a significant increase in the expression of maturation markers 48 h postinfection, along with that of programmed cell death 1 ligand 1 (PD-L1; also called B7-H1 and CD274). JEV-infected DCs expanded the Treg cells in allogenic mixed lymphocyte reactions. The expansion of Treg cells by JEV-infected DCs was significantly reduced upon blocking PD-L1 using an antagonist. In addition, JEV-infected DCs significantly altered the proliferation and reduced the polarization of Th cells toward the Th1-cell phenotype. The results, for the first time, suggest that JEV evades the host's immune system by modulating the crosstalk between DCs and T lymphocytes via the PD-L1 axis.

International audience; High-mobility group box-1 (HMGB1) is remarkably mobile in living cells, which reflects its ability to interact only transiently with both DNA and protein. This property is likely essential for HMGB1 nuclear activities. Nonetheless the weak interaction of HMGB1 with DNA and/or protein partners has also been a major limitation for investigating HMGB1 subnuclear localisation and for the identification of HMGB1 containing complexes by conventional biochemical approaches. In the present study, FRAP experiments demonstrated that DsRed-mediated oligomerization strongly reduces HMGB1 mobility due to an increased affinity for cellular chromatin. Moreover, oligomerized DsRed HMGB1 exhibited a higher affinity for supercoiled DNA in vitro compared to its monomeric counterpart. These results indicate that DsRed-meditated oligomerization is prone to stabilize labile interactions involving HMGB1 both in vivo and in vitro.

Aminoacyl-tRNAs have important roles in a variety of biological processes. Here, we describe the synthesis of stable aminoacyl-tRNA analogs containing 1,4-substituted 1,2,3-triazole rings. The procedure involves (i) copper-catalyzed cycloadditions of 3′-or 2′-azido-adenosine and alkynes, (ii) coupling between the resulting triazole-deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and (iii) the enzymatic ligation of the 2′- or 3′-triazole-dinucleotides with a 22-nt RNA microhelix that mimics the acceptor arm of tRNA. Each nucleoside and nucleotide intermediate was characterized by MS spectrometry and 1H, 31P, and 13C NMR spectroscopy, and the tRNA-analogs were assayed for inhibition of FemXWv, an alanyl-transferase essential for the formation of the peptidoglycan network of Gram-positive bacterial pathogens. The low IC50 values obtained (2 to 4 µM) indicate that the five-membered triazole rings acted as an isosteres of esters and can be used for the design of stable aminoacyl-tRNA analogs. Curr. Protoc. Nucleic Acid Chem. 44:4.44.1-4.44.33. © 2011 by John Wiley & Sons, Inc. Keywords: aminoacyl-tRNA; click chemistry; dinucleotides; inhibitors; triazoles; azidoadenosine; transferase

Background Plant lectins such as Galanthus nivalis agglutinin (GNA) and Hippeastrum hybrid agglutinin (HHA) are natural proteins able to link mannose residues, and therefore inhibit HIV-target cell interactions. Plant lectins are candidate for microbicide development. Objective To evaluate the activity against HIV of the mannose-specific plant lectins HHA and GNA at the cellular membrane level of epithelial cells and monocyte-derived dendritic cells (MDDC), two potential target cells of HIV at the genital mucosal level. Methods The inhibitory effects of HHA and GNA were evaluated on HIV adsorption to genital epithelial HEC-1A cell line, on HIV transcytosis throughout a monolayer of polarized epithelial HEC-1A cells, on HIV adsorption to MDDC and on transfer of HIV from MDDC to autologous T lymphocytes. Results HHA faintly inhibited attachment to HEC-1A cells of the R5-tropic HIV-1Ba-L strain, in a dose-dependent manner, whereas GNA moderately inhibited HIV adsorption in the same context, but only at high drug doses. Only HHA, but not GNA, inhibited HIV-1JR-CSF transcytosis in a dose-dependent manner. By confocal microscopy, HHA, but not GNA, was adsorbed at the epithelial cell surface, suggesting that HHA interacts specifically with receptors mediating HIV-1 transcytosis. Both plant lectins partially inhibited HIV attachment to MDDC. HHA inhibited more efficiently the transfer of HIV from MDDC to T cell, than GNA. Both HHA and GNA lacked toxicity below 200 μg/ml irrespective the cellular system used and do not disturb the monolayer integrity of epithelial cells. Conclusion These observations demonstrate higher inhibitory activities of the lectin plant HHA by comparison to GNA, on HIV adsorption to HEC-1A cell line, HIV transcytosis through HEC-1A cell line monolayer, HIV adsorption to MDDC and HIV transfer from MDDC to T cells, highlighting the potential interest of HHA as effective microbicide against HIV.

Variability in the handling of samples of genital secretions prior to quantitation of HIV-1 RNA and HSV DNA may profoundly affect both the detection and quantitation of these nucleic acids. Over 144 h, we evaluated, the influence of storage temperature (4 degrees C, 20 degrees C, 30 degrees C) on the quantity of HIV-1 RNA and HSV-2 DNA in HIV and HSV negative cervicovaginal lavage pools spiked with known amounts of HIV-1 and HSV-2 and in HIV-1 and HSV-2 co-infected cervicovaginal lavage pools. The level of viral nucleic acids remained stable at 4 degrees C for 24h but decreased significantly when cervicovaginal lavages were stored at 20 degrees C and 30 degrees C, demonstrating that, cervicovaginal lavages to be quantified for viral RNA or DNA require, at minimum, immediate storage at 4 degrees C.

The major peptidoglycan hydrolase of Enterococcus faecalis , AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N -acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.

Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1.

International audience; Heme serves as a prosthetic group of numerous proteins involved in the oxidative metabolism. As result of various pathological conditions associated with hemolysis or tissue damage, large quantities of hemopro-teins and heme can be released extracellularly. Extracellular heme has pronounced pathogenic effects in hemolytic diseases, mediated by its pro-oxidative and pro-inflammatory activities. The pathogenic potential of heme is mostly expressed when the molecule is in protein unbound form. The pathological relevance of free heme deems it necessary to develop reliable approaches for its assessment. Here we developed a technique based on UV-vis absorbance spectroscopy, where cysteine was used as a spec-troscopy probe to distinguish between heme-bound to plasma proteins or hemoglobin from free heme. This technique allowed estimation of the heme-binding capacity of human serum, of particular heme scavenging proteins (albumin, hemopexin) or of immunoglobulins. The main advantage of the proposed approach is that it can distinguish free heme from heme associated with proteins with a wide range of affinities. The described strategy can be used for evaluation of heme-binding capacity of human plasma or serum following intravascular hemolysis or for estimation of stoichiometry of interaction of heme with a given protein.