Dengue virus infection can lead to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) in patients. Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks associated with DHF/DSS patients. However, the mechanisms that cause pathology in DHF/DSS patients are still unclear. Because conversion of plasminogen (Plg) to plasmin (Plm) is the first step in the activation of fibrinolysis, Abs against Plg found in DHF/DSS patients may be important. Therefore, to investigate the specificity, function, and possible origin of these Abs, we generated several Plg cross-reactive mAbs from DENV-immunized mice. An IgG mAb, 6H11, which recognizes an epitope associated with a dengue envelope protein, demonstrated a high level of cross-reactivity with Plg. The 6H11 Ab was further characterized with regard to its effect on Plg activation. Using Plm-specific chromogenic substrate S-2251, we found that mAb 6H11 demonstrated serine protease activity and could convert Plg directly to Plm. The serine protease activity of mAb 6H11 was further confirmed using serine protease chromogenic substrate S-2288. In addition, we found several Plg cross-reactive mAbs that could enhance urokinase-induced Plg activation. Lastly, mAb 6H11 could induce Plm activity and increase the level of D-dimer (a fibrin degradation product) in both human and mouse platelet-poor plasma. Taken together, these data suggest DENV-induced Plg cross-reactive Abs may enhance Plg conversion to Plm, which would be expected to contribute to hyperfibrinolysis in DHF/DSS patients.

Interleukin-17 is a T cell-derived proinflammatory cytokine. This cytokine is suspected to be involved in the development of rheumatoid arthritis (RA) because this cytokine expression is augmented in synovial tissues of RA patients. The pathogenic roles of IL-17 in the development of RA, however, still remain to be elucidated. In this study, effects of IL-17 deficiency on collagen-induced arthritis (CIA) model were examined using IL-17-deficient mice (IL-17(-/-) mice). We found that CIA was markedly suppressed in IL-17(-/-) mice. IL-17 was responsible for the priming of collagen-specific T cells and collagen-specific IgG2a production. Thus, these observations suggest that IL-17 plays a crucial role in the development of CIA by activating autoantigen-specific cellular and humoral immune responses.

IgG antifilaggrin autoantibodies (AFA) are the most specific serological markers of rheumatoid arthritis. In epithelial tissues, they recognize citrulline-bearing epitopes present on various molecular forms of (pro)filaggrin. Histological analysis of rheumatoid synovial membranes with an Ab to citrulline showed labeling of interstitial amorphous deposits and mononuclear cells of various types. Immunochemical analysis of exhaustive sequential extracts of the same tissues showed that they contain several deiminated (citrulline containing) proteins. Among them, two proteins, p64--78 and p55--61, present in urea-DTT and guanidine extracts, were shown by immunoblotting to be specifically targeted by AFA. By amino-terminal sequencing the proteins were identified as deiminated forms of the alpha- and beta-chains of fibrin, respectively. Their identity was confirmed using several Abs specific for the A alpha- and/or to the B beta-chain of fibrin(ogen). Moreover, AFA-positive rheumatoid arthritis (RA) sera and purified AFA were highly reactive to the A alpha- and B beta-chains of human fibrinogen only after deimination of the molecules by a peptidylarginine deiminase. Autoantibodies affinity purified from a pool of RA sera onto deiminated fibrinogen were reactive toward all of the epithelial and synovial targets of AFA. This confirmed that the autoantibodies to the deiminated A alpha-and B beta-chains of fibrinogen, the autoantibodies to the synovial proteins p64--78 and p55--61, and, lastly, AFA, constitute largely overlapping autoantibody populations. These results show that deiminated forms of fibrin deposited in the rheumatoid synovial membranes are the major target of AFA. They suggest that autoimmunization against deiminated fibrin is a critical step in RA pathogenesis.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.

Distinct profiles of autoantibodies directed to intracellular antigens can be detected in the systemic connective tissue diseases. They aid in establishing the correct diagnosis and are included in many sets of diagnostic criteria, such as the ones developed for systemic lupus erythematosus (anti-Smith antigen and anti-double-strand DNA antibodies), mixed connective tissue disease (anti-U1-nuclear ribonucleoprotein antibodies), and Sjögren's syndrome (SS)(anti-SS-A/Ro and anti-SS-B/La antibodies). They are useful prognostic markers in some situations and facilitate clinical and treatment follow-up. Autoantibodies have also been used as probes to gain insights into cell biology, helping to isolate and purify intracellular proteins involved in key cellular functions. We give detailed information on two of the most useful techniques for the detection of autoantibodies in the clinical and research laboratory settings, indirect immunofluorescence and immunoblotting. We also discuss several of the antigen-autoantibody systems found in systemic lupus erythematosus (Smith antigen, U1-nuclear ribonucleoprotein, SS-A/Ro, SS-B/La, proliferating cell nuclear antigen ribosomal ribonucleoprotein, double-strand DNA, histones, antiphospholipids, Ku, Ki/SL), systemic sclerosis (centromere, topo I, RNA polymerases, fibrillarin, polymyositis-Scl, Th/To), polymyositis/dermatomyositis (transferRNA synthetases, signal recognition particle, and others), and SS (SS-A/Ro, SS-B/La, nucleolar organizing region-90, p80-coilin), addressing their clinical significance, common detection methods, immunogenetic associations, and the molecular and cellular biology of the cognate antigens.

Lyn-deficient mice were generated to analyze the role of Lyn in B cell antigen receptor (BCR) signaling. These mice had a reduced number of peripheral B cells with a greater proportion of immature cells and a higher than normal turnover rate. Aged lyn-/- mice developed splenomegaly, produced autoantibodies, and had an expanded population of B lymphoblasts of the B1 lineage. Splenic B cells from young lyn-/- mice initiated early BCR signaling events, although in a delayed fashion. Unexpectedly, lyn-/- B cells exhibited an enhanced MAP kinase activation and an increased proliferative response to BCR engagement. Stimulation of lyn-/- B cells with intact and F(ab')2 anti-IgM revealed defects in at least two mechanisms that negatively regulate BCR signaling, one of which involves Fc gammaRIIb1.

Atherosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This "oxidation hypothesis" has gained general acceptance in recent years, and evidence for the role of lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E-deficient mice (apo E-/-/L-12LO-/-). In chow-fed 15-week-old apo E-/-/L-12LO-/- mice, the extent of lesions in whole-aorta en face preparations (198 +/- 60 microm2) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-lipoxygenase-expressing controls (apo E-/-/L-12LO+/+), which showed areas of lipid deposition (15,700 +/- 2,688 microm2) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E-deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E-/-/L-12LO-/- mice. The combined data indicate a role for 12/15-lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

Antifilaggrin autoantibodies (AFA) are a population of IgG autoantibodies associated to rheumatoid arthritis (RA), which includes the so-called "antikeratin" Abs and antiperinuclear factor. AFA are the most specific serological markers of RA. We previously showed that they recognize human epidermal filaggrin and other profilaggrin-related proteins of various epithelial tissues. Here, we report further characterization of the protein Ags and epitopes targeted by AFA. All the Ags that exhibit numerous neutral/ acidic isoelectric variants were immunochemically demonstrated to be deiminated proteins. In vitro deimination of a recombinant human filaggrin by a peptidylarginine deiminase generated AFA epitopes on the protein. Moreover, two of three filaggrin-derived synthetic peptides with a citrulline in the central position were specifically and widely recognized by AFA affinity-purified from a series of RA sera. These results indicate that citrulline residues are constitutive of the AFA epitopes, but only in the context of specific amino acid sequences of filaggrin. In competition experiments, the two peptides abolished the AFA reactivity of RA sera, showing that they present major AFA epitopes. These data should help in the identification of a putative deiminated AFA-inducing or cross-reactive articular autoantigen and provide new insights into the pathogenesis of RA. They could also open the way toward specific immunosuppressive and/or preventive therapy of RA.

A human thyroid cDNA library was screened by hybridization with a dog thyrotropin receptor (TSHr) cDNA. Sequencing of the resulting clones identified a 2292 residue open reading frame encoding a 744 amino acid mature polypeptide presenting 90.3% similarity with the dog TSHr. Two major transcripts (4.6 and 4.4 kilobases) were identified in the human thyroid which suggests that alternative splicing could generate multiple forms of human TSHr. Transfection of the coding sequence in COS-7 cells conferred to a membrane preparation of these cells the ability to bind specifically TSH. TSH binding was completely displaced by immunoglobulin preparations from patients with idiopathic myxoedema.

We recently cloned monoclonal IgM autoantibodies which bind to epitopes of oxidized low-density lipoprotein (OxLDL) from apoE-deficient mice (EO- autoantibodies). We now demonstrate that those EO- autoantibodies that were originally selected for binding to copper-oxidized low-density lipoproteins (CuOx-LDL), also bound both to the oxidized protein and to the oxidized lipid moieties of CuOx-LDL. The same EO- autoantibodies showed specific binding to products of oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine (OxPAPC) and to the specific oxidized phospholipid, 1-palmitoyl-2-(5-oxovaleroyl)-phosphatidyl-choline (POVPC), whereas oxidation of fatty acids (linoleic or arachidonic acid) or cholesteryl esters (cholesteryl-oleate or cholesteryl-linoleate) did not yield any binding activity. Those EO- autoantibodies that bound to oxidized phospholipids (e.g., EO6) inhibited the binding and degradation of CuOx-LDL by mouse peritoneal macrophages up to 91%, whereas other IgM EO- autoantibodies, selected for binding to malondialdehyde (MDA)-LDL, had no influence on binding of either CuOx-LDL or MDA-LDL by macrophages. F(ab')2 fragments of EO6 were equally effective as the intact EO6 in preventing the binding of CuOx-LDL by macrophages. The molar ratios of IgM to LDL needed to maximally inhibit the binding varied from approximately 8 to 25 with different CuOx-LDL preparations. Finally, a POVPC-bovine serum albumin (BSA) adduct also inhibited CuOx-LDL uptake by macrophages. These data suggest that oxidized phospholipid epitopes, present either as lipids or as lipid-protein adducts, represent one class of ligands involved in the recognition of OxLDL by macrophages, and that apoE-deficient mice have IgM autoantibodies that can bind to these neoepitopes and inhibit OxLDL uptake.