Ikaros Is a Negative Regulator of B1 Cell Development and Function.

B1 B cells secrete most of the circulating natural antibodies and are considered key effector cells of the innate immune response. However, B1 cell-associated antibodies often cross-react with self-antigens, which leads to autoimmunity, and B1 cells have been implicated in cancer. How B1 cell activity is regulated remains unclear. We show that the Ikaros transcription factor is a major negative regulator of B1 cell development and function. Using conditional knock-out mouse models to delete Ikaros at different locations, we show that Ikaros-deficient mice exhibit specific and significant increases in splenic and bone marrow B1 cell numbers, and that the B1 progenitor cell pool is increased ∼10-fold in the bone marrow. Ikaros-null B1 cells resemble WT B1 cells at the molecular and cellular levels, but show a down-regulation of signaling components important for inhibiting proliferation and immunoglobulin production. Ikaros-null B1 cells hyper-react to TLR4 stimulation and secrete high amounts of IgM autoantibodies. These results indicate that Ikaros is required to limit B1 cell homeostasis in the adult.

Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.

[Pharyngeal fibrovascular polyp]. / Polype fibrovasculaire de l'hypopharynx.

The giant fibrovascular polyp of the hypopharynx is a rare and benign tumor. We report one case in a 46-year-old man. This lesion, usually unique, affects predominantly men with an average age of 53 years. These polyps are located predominantly in the upper esophagus and rarely in the hypopharynx. They are usually asymptomatic and small, detected by endoscopy. They can grow to considerable length and cause digestive or respiratory symptoms. Histologically, fibrovascular polyps consist of a various mixture of fibrous and lipomatous elements with abundant vascularisation and they are covered by normal squamous epithelium. The lack of muscular support and the pressure difference in the peristaltic wave contribute to polyp formation.

Heteroclitic properties of mixed alpha- and aza-beta3-peptides mimicking a supradominant CD4 T cell epitope presented by nucleosome.

Recent studies have revealed that peptide analogues containing modified peptide bonds might replace poorly stable natural peptides in therapeutic strategies. Using the model peptide 88-99 of histone H4, which contains a supradominant epitope recognized by Th cells induced to nucleosomes, we have generated twelve analogues containing aza-beta(3)-amino acid residue substitutions. The ability of this new class of peptidomimetics corresponding to the Psi[CONHNRCH(2)] modification to be recognized by T cells primed with the parent peptide was examined in BALB/c mice. An Ala-scan study revealed that residues 88 to 92 were essential for keeping antigenic activity of the nominal peptide. In good agreement, the six aza-beta(3)-analogues encompassing substitutions in the region 89-92 were antigenically inactive. Analogues PsiG94 and PsiG99 were both antigenic and immunogenic, though at levels that were slightly lower to that of the parent peptide. However, the remaining analogues PsiR95, PsiL97, PsiY98 and PsiL97-Y98 were strongly recognized by T cells generated to the homologous peptides. The PsiL97-Y98 analogue, in particular, strongly activated CD4(+) T cells as visualized in CFSE dilution assay. T cells primed to these four analogues and recalled with the nominal peptide secreted high levels of either IL-2 (PsiR95, PsiY98) or IFN-gamma (PsiL97, PsiL97-Y98). This result, supported by molecular modeling, suggests that TCRs of T cells primed to these four analogues recognized the parent peptide associated with the MHC I-A(d)/I-E(d) molecules. Since these T cells produce a distinct cytokine pattern when they are recalled with the parent sequence, this new class of analogues may have valuable applications in the context of self-tolerance and autoimmunity.

52-kDa Ro/SSA epitopes preferentially recognized by antibodies from mothers of children with neonatal lupus and congenital heart block.

Neonatal lupus erythematosus is a rare disorder caused by the transplacental passage of maternal autoantibodies. The 52-kDa Ro/SSA antigen (Ro52) ribonucleoprotein represents an antigenic target strongly associated with the autoimmune response in mothers whose children have neonatal lupus and cardiac conduction disturbances, mainly congenital heart block. The objective of this study was to identify putative Ro52/60-kDa Ro/SSA antigen (Ro60) epitopes associated with neonatal lupus and congenital heart block. The reactivity of IgG antibodies present in the sera from mothers with systemic lupus erythematosus and Sjögren's syndrome and in the sera from asymptomatic mothers (a longitudinal study of 192 samples from 66 subjects) was investigated by ELISA using Ro52, Ro60 and 48-kDa La/SSB antigen proteins, as well as 45 synthetic peptides, 13-24 residues long, of Ro52/Ro60 proteins. One to 19 samples collected before, during and after pregnancy were available for each mother. Forty-three disease controls selected randomly and normal sera were tested in parallel. Although no differences were found between Sjögren's syndrome and asymptomatic mothers of group I, who had at least one infant with neonatal lupus, and of group II, who had healthy babies only, significant differences were observed between lupus mothers from both groups. In the former group of lupus mothers, a significantly higher frequency of antibodies to Ro52 peptides 107-122 and 277-292 was observed. Between 18 and 30 weeks of gestation, the period of risk, there was clearly an elevated level of antibodies reacting with Ro52 peptides 1-13, 277-292 and 365-382. Antibodies to Ro52 peptide 365-382 have been shown previously to cross-react with residues 165-185 of the heart 5-HT4 serotoninergic receptor, and might be pathologically important. The level of these Ro52 antibody subsets decreased at the end of pregnancy and after delivery. IgG antibodies to Ro52 peptides 1-13, 107-122, 277-292 and 365-382 may therefore represent important biomarkers to predict a complication in pregnant lupus women with Ro52 antibodies.

Solid-phase synthesis of "mixed" peptidomimetics using Fmoc-protected aza-beta3-amino acids and alpha-amino acids.

[structure: see text] A solid-phase fluorenylmethyloxycarbonyl (Fmoc)-based synthesis strategy is described for "mixed" aza-beta3-peptides as well as a convenient general approach for their required building blocks, the aza-beta3-amino acid residues (aza-beta3-aa). These monomers allow the synthesis of relatively large quantities of pure mixed aza-beta3-peptides. The required Fmoc-substituted aza-beta3-amino acids are accessible by convenient synthesis, and a number of monomers including those containing side chains with functional groups have been synthesized. The method was applied toward the solid-phase synthesis of aza-beta3-peptide mimetics of a biologically active histone H4 sequence.

The caveolin-1 binding domain of HIV-1 glycoprotein gp41 is an efficient B cell epitope vaccine candidate against virus infection.

Caveolin-1 is a scaffolding protein that organizes and concentrates specific ligands within the caveolae membranes. We identified a conserved caveolin-1 binding motif in the HIV-1 transmembrane envelope glycoprotein gp41 and designed several synthetic peptides, referred to as CBD1, corresponding to the consensus caveolin-1 binding domain in gp41. In rabbits, these peptides elicit the production of antibodies that inhibit infection of primary CD4(+) T lymphocytes by various primary HIV-1 isolates. Interestingly, gp41 exists as a stable complex with caveolin-1 in HIV-infected cells. Anti-CBD1 peptide antibodies, therefore, might be functional by inhibiting the potential interaction of gp41 with caveolin-1. Because of their capacity to elicit antibodies that inhibit the different clades of HIV-1, CBD1-based peptides may represent a novel synthetic universal B cell epitope vaccine candidate for HIV/AIDS. Moreover, such peptides could also have an application as a therapeutic vaccine since CBD1-specific antibodies are rare in HIV-infected individuals from several geographic origins.

CD4+ T cells from (New Zealand Black x New Zealand White)F1 lupus mice and normal mice immunized against apoptotic nucleosomes recognize similar Th cell epitopes in the C terminus of histone H3.

We have previously reported that peptide 88-99 of histone H4 represents a minimal T cell epitope recognized by Th cells from nonautoimmune BALB/c (H-2(d/d)) mice immunized with nucleosomes. In this study, we tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 for recognition by CD4(+) T cells from unprimed (New Zealand Black (NZB) x New Zealand White (NZW))F(1) lupus mice (H-2(d/z)). None of the 11 H4 peptides was recognized by CD4(+) T cells from (NZB x NZW)F(1) mice. In contrast, these cells proliferated and secreted IL-2, IL-10, and IFN-gamma upon ex vivo stimulation with H3 peptides representing sequences 53-70, 64-78, and 68-85. Peptides 56-73 and 61-78 induced the production of IFN-gamma and IL-10, respectively, without detectable proliferation, suggesting that they may act as partial agonist of the TCR. Th cells from unprimed BALB/c mice and other lupus-prone mice such as SNF(1) (H-2(d/q)) and MRL/lpr (H-2(k/k)) mice did not recognize any peptides present within the H3 region 53-85. We further demonstrated that immunization of normal BALB/c mice with syngeneic liver nucleosomes and spleen apoptotic cells, but not with nonapoptotic syngeneic cells, induced Th cell responses against several peptides of the H3 region 53-85. Moreover, we found that this conserved region of H3, which is accessible at the surface of nucleosomes, is targeted by Abs from (NZB x NZW)F(1) mice and lupus patients, and contains motifs recognized by several distinct HLA-DR molecules. It might thus be important in the self-tolerance breakdown in lupus.

Specificity and effect on apoptosis of Tat antibodies from vaccinated and SHIV-infected rhesus macaques and HIV-infected individuals.

Recent contributions have demonstrated that actively secreted Tat protein plays an important functional role in human immunodeficiency virus-1 (HIV-1) infection and that Tat antibodies might interfere with disease progression by blocking the protein extracellularly. In this context we have studied the recognition of several Tat mutants as well as various synthetic Tat fragments by anti-Tat monoclonal antibodies and by IgG antibodies from a large collection of slow and fast-progressor infected individuals. We have also tested the sera from simian/human immunodeficiency virus (SHIV)-infected macaques with these Tat peptides. Important differences were found between long-term non-progressors and fast-progressors, and between human and monkey sera in terms of antibody specificity. Rabbits and macaques were immunised with several Tat peptides and we found that certain antibody subsets from immunised animals recognised the cognate protein Tat and had the capacity to inhibit Tat-induced apoptosis of T cells. Such antibodies might be important for controlling Tat-induced death in cells uninfected by HIV-1.

Structural and immunological characterisation of heteroclitic peptide analogues corresponding to the 600-612 region of the HIV envelope gp41 glycoprotein.

The conformational and immunological properties of different analogues corresponding to the 600-612 disulfide loop of the human immunodeficiency virus (HIV) gp41 glycoprotein envelope were studied. Fourteen analogues were designed and synthesised; namely, a series of seven analogues in which the disulfide bond was replaced by a lactam bridge and a series of seven analogues in which one residue of each analogue at a time, was replaced by its corresponding homologised alpha-amino acid (beta(3)-amino acid). In the case of the lactam analogues, the influence of the two possible CO-NH and NH-CO orientations of the lactam bridge as well as the size of the lactam ring was explored. The analogues were tested in ELISA with monoclonal antibodies raised against the 600-612 cyclic parent peptide as well as with sera from HIV-1 infected patients. A structural analysis of the parent and analogue peptides was carried out in dimethyl sulfoxide (DMSO-d(6)) using two-dimensional NMR techniques and molecular dynamics simulations. Comparison of the own conformation of the cyclic analogues with their either strong or weak reactivity with the antibodies reveals structural features that may be correlated with the antibody reactivity. Thus, a close structural similarity, particularly a characteristic orientation of the side-chains of residues Lys606, Leu607 and Ile608 in the loop, was found in certain beta(3)-analogues that were better recognised than the parent peptide by anti-peptide mouse monoclonal antibodies and patients' antibodies.