Patterns of IgE sensitization in house dust mite-allergic patients: implications for allergen immunotherapy.

BACKGROUND: Understanding patterns of IgE sensitization in Dermatophagoides-allergic patients living in various geographical areas is necessary to design a product suitable for worldwide allergen immunotherapy (AIT). METHODS: Using a HIFI Allergy customized microarray assay, IgEs specific for 12 purified allergens from Dermatophagoides pteronyssinus or D. farinae were assessed in sera from 1302 house dust mite (HDM)-allergic patients living in various areas. Comprehensive mass spectrometric (MS) analyses were conducted to characterize HDM extracts, as well as purified bodies and feces. RESULTS: Patterns of IgE reactivity to HDM allergens are comparable in all cohorts of patients analyzed, encompassing adults and 5- to 17-year-old children, as well as American, Canadian, European, and Japanese patients. Overall, >70% and >80% of HDM-allergic patients are sensitized to group 1 and group 2 allergens, respectively, from D. pteronyssinus and/or D. farinae species. Furthermore, 20-47% of patients also have IgEs to allergens from groups 4, 5, 7, 13, 15, 21, and 23. All patients have IgEs to allergens present in mite bodies and feces. MS-based analyses confirmed the presence of mite allergens recorded by IUIS in D. pteronyssinus and D. farinae extracts, with groups 2, 8, 10, 11, 14, and 20 prominent in bodies and groups 1, 6, 18, and 23 well represented in feces. CONCLUSIONS: Mite-specific AIT should rely upon a mixture of D. pteronyssinus and D. farinae extracts, manufactured from both feces and bodies. Such a combination is appropriate to treat children and adult Dermatophagoides-allergic patients from Asia, Europe, and North America.

Heterogeneity of antibody responses among clinical responders during grass pollen sublingual immunotherapy.

BACKGROUND: During allergen-specific sublingual immunotherapy (SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. OBJECTIVE: To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. METHODS: Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. RESULTS: A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level.

Advances in the quantification of relevant allergens in allergenic extracts.

Relevant allergens are major contributors to the safety and efficacy of allergenic extracts used in allergen immunotherapy (AIT). As such, they should be accurately quantified, as recommended by the 2008 European guidelines on allergen products. Until now, the quantification of relevant allergens was mainly performed by using immunoassays (e.g. ELISA) that relying upon specific antibodies. Although antibody-based quantification is commonly used to assess the concentration of relevant allergens in allergenic extracts, results must be taken with caution in the light of the inherent limitations of such techniques. In the present study, we discuss how those limitations can be overcome by using comprehensive mass spectrometry-based techniques.

Molecular variability of group 1 and 5 grass pollen allergens between Pooideae species: implications for immunotherapy.

BACKGROUND: Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. METHODS: Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II-restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)-binding assays. CD4+ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. RESULTS: MS analysis of group 5 pollen allergens reveals considerable intra- and inter-species variability in amino acid sequence, with 30-50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N- and O-glycosylation contribute to the variability of group 1 allergens, yielding 5-10 main isoforms, depending on the species. Out of 14 MHC class II-restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA-DR molecules result in variable CD4+ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species-restricted (or semi-restricted) epitopes associated with group 1 or 5 allergens, respectively. CONCLUSION: Major pollen allergens from distinct grass species bear both shared and species-restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized.

Targeting the allergen to oral dendritic cells with mucoadhesive chitosan particles enhances tolerance induction.

BACKGROUND: Sublingual immunotherapy (SLIT) efficacy could be improved by formulations facilitating allergen contact with the oral mucosa and uptake by antigen-presenting cells (APCs). METHODS: Two types of chitosan microparticles, differing in size and surface charge, were tested in vitro for their capacity to improve antigen uptake and presentation by murine bone marrow-derived dendritic cells (BMDCs) or purified oral APCs. T-cell priming in cervical lymph nodes (LNs) was assessed by intravenous transfer of carboxyfluorescein diacetate succinimidyl ester-labelled ovalbumin (OVA)-specific CD4+ T cells and flow cytometry analysis. Ovalbumin-sensitized BALB/c mice were treated sublingually with soluble or chitosan-formulated OVA twice a week for 2 months. Airway hyperresponsiveness (AHR), lung inflammation and T-cell responses in cervical and mediastinal LNs were assessed by whole-body plethysmography, lung histology and Cytometric Bead Array technology, respectively. RESULTS: Only a mucoadhesive (i.e. highly positively charged) and microparticulate form of chitosan enhances OVA uptake, processing and presentation by murine BMDCs and oral APCs. Targeting OVA to dendritic cells with this formulation increases specific T-cell proliferation and IFN-gamma/IL-10 secretion in vitro, as well as T-cell priming in cervical LNs in vivo. Sublingual administration of such chitosan-formulated OVA particles enhances tolerance induction in mice with established asthma, with a dramatic reduction of both AHR, lung inflammation, eosinophil numbers in bronchoalveolar lavages, as well as antigen-specific Th2 responses in mediastinal LNs. CONCLUSIONS: Mucoadhesive chitosan microparticles represent a valid formulation for sublingual allergy vaccines.

Standardization of allergen products: 1. Detailed characterization of GMP-produced recombinant Bet v 1.0101 as biological reference preparation.

BACKGROUND: Standardization of allergen extracts requires the availability of well-characterized recombinant allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. METHODS: Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. RESULTS: Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4 degrees C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. CONCLUSION: Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1.

Production of native and modified recombinant Der p 1 molecules in tobacco plants.

BACKGROUND: As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. OBJECTIVE: Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N-glycosylation sites (Gly(-)) and/or cysteine protease activity (Enz(-)). Methods Using Agrobacterium tumefaciens-based transformation, pro Der p 1 molecules bearing mutations within either the N-glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. RESULTS: Four forms of recombinant Der p 1 (i.e. wild-type Gly(+)/Enz(+), as well as Gly(-)/Enz(+), Gly(+)/Enz(-) or Gly(-)/Enz(-) variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly(-)/Enz(-) variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly(+)/Enz(+) form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. CONCLUSION: A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.

Toll-like receptor 2 agonist Pam3CSK4 enhances the induction of antigen-specific tolerance via the sublingual route.

BACKGROUND: Sublingual immunotherapy (SLIT) has been established in humans as a safe and efficacious treatment for type I respiratory allergies. OBJECTIVE: In this study, we compared three Toll-like receptor (TLR) 2 ligands (Pam3CSK4, Porphyromonas gingivalis lipopolysaccharide and lipoteichoic acid) as potential adjuvants for sublingual allergy vaccines. METHODS: These molecules were tested in co-cultures of adjuvant-pre-treated dendritic cells (DCs) with murine naïve CD4(+) T lymphocytes. Patterns of cytokine production, phenotype, proliferation and gene expression were analysed by ELISA, cytofluorometry and quantitative PCR, respectively. TLR2 ligands were subsequently tested in a model of SLIT in BALB/c mice sensitized with ovalbumin (OVA). RESULTS: Among the three TLR2 ligands tested, the synthetic lipopeptide Pam3CSK4 is the most potent inducer of IL-12p35 and IL-10 gene expression in murine bone marrow-derived DCs, as well as in purified oral myeloid DCs. Only Pam3CSK4-treated DCs induce IFN-gamma and IL-10 secretion by naïve CD4(+) T cells. Sublingual administration of Pam3CSK4 together with the antigen in BALB/c mice sensitized to OVA decreases airway hyperresponsiveness as well as OVA-specific T-helper type 2 (Th2) responses in cervical lymph nodes dramatically. CONCLUSION: Pam3CSK4 induces Th1/regulatory T cell responses, and as such, is a valid candidate adjuvant for sublingual allergy vaccines.

Presence of antinucleosome autoantibodies in a restricted set of connective tissue diseases: antinucleosome antibodies of the IgG3 subclass are markers of renal pathogenicity in systemic lupus erythematosus.

OBJECTIVE: To study the frequency and disease specificity of antinucleosome antibody reactivity in diverse connective tissue diseases (CTD), and to determine factors, such as antibody subclass, that may influence the pathogenicity of these antibodies in relation to disease activity. METHODS: IgG and IgM antinucleosome activities on nucleosome core particles from 496 patients with 13 different CTD and 100 patients with hepatitis C were measured by enzyme-linked immunosorbent assay (ELISA). Of the patients with CTD, 120 had systemic lupus erythematosus (SLE), 37 had scleroderma (systemic sclerosis; SSc), 20 had mixed connective tissue disease (MCTD), and 319 had other CTD, including Sjögren's syndrome, inflammatory myopathy, rheumatoid arthritis, primary antiphospholipid syndrome, Wegener's granulomatosis, Takayasu arteritis, giant cell arteritis, relapsing polychondritis, Behçet's syndrome, and sarcoidosis. Antinucleosome-positive sera were further analyzed, by isotype-specific ELISA, for antinucleosome and anti-double-stranded DNA (anti-dsDNA) IgG subclasses. RESULTS: SLE, SSc, and MCTD were the only 3 CTD in which antinucleosome IgG were detected (71.7%, 45.9%, and 45.0% of patients, respectively). Antinucleosomes of the IgG3 subclass were present at high levels in patients with active SLE and were virtually absent in those with SSc, MCTD, or inactive SLE, and their levels showed a positive correlation with SLE disease activity. Of note, an increase in levels of antinucleosome of the IgG3 isotype was observed during SLE flares, and this increase was found to be closely associated with active nephritis. Levels of antinucleosome of the IgG1 subclass showed a trend toward an inverse correlation with SLE disease activity. No significant fluctuation in the anti-dsDNA isotype profile was observed in relation to SLE severity or clinical signs. CONCLUSION: Our data suggest that IgG antinucleosome is a new marker that may help in the differential diagnosis of CTD; antinucleosome of the IgG3 isotype might constitute a selective biologic marker of active SLE, in particular, of lupus nephritis.

Early anti-nucleosome autoantibodies from a single MRL+/+ mouse: fine specificity, V gene structure and pathogenicity.

In systemic lupus erythematosus, the nucleosome assumes a central role in the autoimmune response to self antigens. To gain insight into the etiology and pathogenesis of anti-nucleosome antibodies (Ab), we analyzed a panel of six IgG-secreting hybridomas derived from a single young MRL +/+ mouse at the onset of the autoimmune response. All monoclonal antibodies (mAb) bound exclusively the native nucleosome, and represented five different clonotypes that recognized diverse nucleosomal epitopes, typical of a polyclonal response. The VH-complementarity-determining region (CDR)3 regions exhibited unique stretches of charged amino acids with different polarity that may be important for the interaction with the nucleosome. These early anti-nucleosome mAb displayed striking structural differences with not only anti-DNA, but also with anti-nucleosome Ab, that appear later in disease. Two of the mAb deposited in kidney glomeruli after in vivo administration to RAG-1-deficient mice, suggesting that diverse B cell clones, possibly selected by the nucleosome itself, may play a role in the initiation of kidney damage.

Anticardiolipin, anti-beta2-glycoprotein I, and antinucleosome antibodies in hepatitis C virus infection and mixed cryoglobulinemia. Multivirc Group.

OBJECTIVE: To investigate anticardiolipin antibodies (aCL), anti-beta2-glycoprotein I (anti-beta2GPI), and antinucleosome antibodies in patients with hepatitis C virus (HCV) with or without mixed cryoglobulinemia. aCL can appear in infectious diseases, but are not then associated with thrombotic events. Antibodies directed to beta2GPI, a co-factor of aCL, have been said to be associated with the presence of "autoimmune" aCL. About 50% of cases of essential mixed cryoglobulinemia are associated with HCV infection. High prevalence of autoantibodies directed to nuclear antigens has been found in HCV infection but the prevalence of antibody to nucleosome has not yet been reported. METHODS: Group 1: 29 patients with chronic HCV infection and mixed cryoglobulinemia. Group 2: 17 patients with chronic HCV infection but without mixed cryoglobulinemia. To analyze the possible effect of mixed cryoglobulinemia itself on aCL production, we also studied 22 patients with essential mixed cryoglobulinemia and no HCV infection (Group 3). In addition, 96 healthy blood donors were used as a control group (Group 4). Mixed cryoglobulinemia was detected by immunofixation. Anti-HCV antibodies were detected by 3rd generation tests, aCL, anti-beta2GPI, and antinucleosorne antibodies were detected by ELISA. In patients with mixed cryoglobulinemia, we also looked for aCL separately in cryoprecipitate and in serum after extraction of mixed cryoglobulins to investigate a possible "capture" of aCL in the cryoprecipitate. RESULTS: IgG aCL were more frequently found in patients with HCV than in controls [9/46 (20%) vs 2/96 (2%); p < 0.001]. The prevalence of aCL was similar in patients with HCV with or without mixed cryoglobulinemia (6/29 vs 3/17; p = NS). No patient with positive aCL had anti-beta2GPI, antinucleosome antibodies, thrombotic events, or thrombocytopenia. IgG aCL were more frequent in patients with mixed cryoglobulinemia, whatever their status for HCV infection, than in subjects without mixed cryoglobulinemia [8/51 (16%) vs 5/113 (4%); p < 0.02]. The prevalence of aCL was similar in patients with type II or type III mixed cryoglobulinemia. When we looked for aCL separately in serum and in cryoprecipitate, we did not find aCL in cryoprecipitate. In patients with HCV, the prevalence of aCL was not different whether patients were treated or not with interferon alpha. CONCLUSION: IgG aCL are frequently found in patients with HCV regardless of status for mixed cryoglobulinemia. These aCL have the characteristics of infection related aCL: low titer, absence of thrombotic events, and absence of anti-beta2GPI. The high proportion of aCL in patients with mixed cryoglobulinemia compared to those without, and the absence of antinucleosome antibodies, suggest that these aCL may be secondary to endothelial damage induced by mixed cryoglobulinemia or HCV itself, rather than to nonspecific polyclonal lymphocyte activation.

Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: correlation with serum antinucleosome antibody titers and absence of clear association with disease activity.

OBJECTIVE: To assess nucleosome plasma levels in patients with systemic lupus erythematosus (SLE) and to study the correlations with serum antinucleosome, anti-double-stranded DNA (anti-dsDNA), and antihistone antibody activities, as well as with disease activity (by the SLE Disease Activity Index [SLEDAI]). METHODS: In a cross-sectional study, we assessed 58 SLE patients for their plasma nucleosome levels. Plasma nucleosome levels as well as serum antinucleosome, anti-double-stranded DNA, and antihistone antibody activities were assessed by enzyme-linked immunosorbent assay. SLE activity was evaluated using the SLEDAI: RESULTS: The mean (+/-SD) plasma nucleosome concentration in SLE patients was 52 +/- 159 ng/ml (range 5-1,180), and was significantly higher than that of the controls (16 +/- 8.8 ng/ml, range 8-52; P = 0.03). Thirteen of the 58 lupus patients had levels over the range of normal (defined as the control mean + 3 SD, or 42 ng/ml). An inverse correlation was found between nucleosome plasma levels and serum antinucleosome antibody activity in the entire group of SLE patients, those with active disease, and those with inactive disease, respectively. No correlation was found between the SLEDAI and nucleosome plasma concentrations. CONCLUSION: Nucleosome plasma levels may be normal or increased in SLE, and found in patients with active or inactive SLE. Longitudinal studies are needed to further establish whether high levels of circulating nucleosomes may predict the occurrence of an SLE flare.

Marked depletion at the late pro-B cell stage in the bone marrow of lpr mice correlates with the development of lymphadenopathy but not autoimmunity.

We have found that old lpr mice exhibit a loss of mature B cells in the bone marrow. The deleted population is HSAlo B220hi and is generated from the peripheral pool. Abnormalities in the microenvironment could explain the absence of mature B cells. Thus, old lpr bones were grafted under the skin of normal adult Ly5.1 hosts and examined 3 weeks later for the presence of Ly5.1+ B220hi cells. Our data show that the lpr medullary compartment was efficiently restored by host B cells. These results suggest that the bone marrow microenvironment of old lpr mice is able to sustain mature B cells. However, transfer of T cell-depleted bone marrow cells from old lpr mice to Rag-2 -/- mice leads to incomplete and inefficient repopulation of the host medullary compartment. Thus, a defect at an early stage of B cell differentiation was detected: using four-color flow cytometry, we found a profound depletion of the late pro-B B220+ CD43+ HSA+ BP-1+ cell population in aging lpr mice. This depletion was not observed in old autoimmune-prone MRL-+/+ mice which develop only autoantibodies but was present in B6-lpr mice which develop a lymphadenopathy and an indolent autoimmune syndrome. Altogether, our results demonstrate an age-linked defect in the progression of B cell differentiation in lpr mice independent of the presence of autoantibodies and targeted to the late pro-B cell subset.

Urtica dioica agglutinin, a V beta 8.3-specific superantigen, prevents the development of the systemic lupus erythematosus-like pathology of MRL lpr/lpr mice.

The V beta 8.3-specific superantigenic lectin Urtica dioica agglutinin (UDA) was used to delete the V beta 8.3+ T cells in MRL lpr/lpr mice. In contrast to the systemic lupus erythematosus-like pathology which progresses with age in the phosphate-buffered saline-injected MRL lpr/lpr controls, UDA-treated animals did not develop overt clinical signs of lupus and nephritis. The pathogenic T cell clones thus reside within the V beta 8.3+ T cell population, which includes an expanded T cell clone described previously. Finally, UDA alters the production of autoantibodies in a sex-dependent manner.

Binding of nucleosomes to a cell surface receptor: redistribution and endocytosis in the presence of lupus antibodies.

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds) DNA and/or anti-histone autoantibodies could recognize and influence the fate of cell surface-bound nucleosomes. 125I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of approximately 7nM and a low-affinity site (Kd approximately 400 nM) with a high capacity of 9 x 10(7) sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived ds DNA (180-200 bp) was found to complete for binding of 125I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from 125-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa ds DNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-ds DNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5-10 min which moved toward the perinuclear region by 20-30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the microfilament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-ds DNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.

Presence of nucleosome-restricted antibodies in patients with systemic lupus erythematosus.

OBJECTIVE: To assess whether nucleosome-restricted antibodies, i.e., antibodies that react with the whole nucleosome particle but not with its individual components (double-stranded DNA [dsDNA] and histones), are present in the sera of patients with systemic lupus erythematosus (SLE). METHODS: Antibodies were detected by enzyme-linked immunosorbent assay using purified nucleosomes, dsDNA, or histones. These tests were applied to the sera of 40 patients with SLE. Protein G-purified IgGs of representative sera were sequentially adsorbed on dsDNA- and histone-conjugated solid-phase supports and further assayed for their nucleosome, dsDNA, and histone reactivities. RESULTS: Of the 40 sera tested, 16 displayed anti-dsDNA and/or antihistone antibody activity, which was always associated with significant antinucleosome reactivity. In addition, 3 sera showed antinucleosome activity that was not associated with concomitant anti-dsDNA or antihistone activity. The presence of true nucleosome-restricted antibodies was demonstrated, after solid-phase adsorption, in representative SLE sera that showed anti-dsDNA or antihistone antibody activity, and also in sera that did not show these activities. CONCLUSION: Our results provide evidence for the presence of nucleosome-restricted antibodies in patients with lupus. These nucleosome-restricted antibodies, along with anti-dsDNA and antihistone antibodies, appear to belong to a broad set of antinuclear antibodies, the antinucleosome family.

Nucleosome-restricted antibodies are detected before anti-dsDNA and/or antihistone antibodies in serum of MRL-Mp lpr/lpr and +/+ mice, and are present in kidney eluates of lupus mice with proteinuria.

OBJECTIVE: To compare the humoral response to nucleosomes with the response to their individual components (double-stranded DNA [dsDNA] and histones) and to assess the involvement of antinucleosome antibodies in immune deposits in the kidney of MRL mice. METHODS: We used enzyme-linked immunosorbent assays of sera and kidney eluates for antibody activity against purified nucleosomes, dsDNA, and histones. RESULTS: Antinucleosome antibodies emerged before anti-dsDNA and antihistone antibodies. A fraction of antinucleosome antibodies reacted exclusively with nucleosomes and not with their components, dsDNA and histones. These nucleosome-restricted antibodies were detected in the proteinuric MRL mouse kidney eluate. CONCLUSION: Our findings support the notion that nucleosomes play a major role in the emergence of antinuclear autoantibodies and that antinucleosome antibodies might be involved in the nephritogenic process in murine lupus.

[The appearance of antinucleosome antibodies precedes that of anti-double-stranded DNA and anti-histones in murine lupus]. / L'apparition des anticorps anti-nucléosome précède celle des anti-DNA double brin et des anti-histones au cours du lupus murin.

Collected samples serum of 30 MRL lpr/lpr and 15 MRL MP +/+ were tested by ELISA on purified mononucleosomes and its individual components, dsDNA and histones. Anti-mononucleosome antibodies can be detected as early as 10 and 23 week of age for MRL lpr/lpr and MP +/+, respectively, and arose before anti-dsDNA and anti-histone antibodies.