AM3, a natural glycoconjugate, induces the functional maturation of human dendritic cells.

BACKGROUND AND PURPOSE: Dendritic cells (DCs) are dedicated antigen-presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen-specific T-lymphocyte responses. Here, we have investigated the effects of Inmunoferon-active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte-derived DCs (MDDCs). EXPERIMENTAL APPROACH: MDDCs derived from healthy and hepatitis C virus (HCV)-infected patients were stimulated with AM3. We analysed the expression of cell surface proteins by flow cytometry, that of cytokine production by ELISA, and the expression of chemokines and chemokine receptors by RNase protection assays. T-lymphocyte proliferation was assessed in mixed lymphocyte reactions, protein expression by western blot and luciferase-based reporter methods, and Toll-like receptor (TLR)-blocking antibodies were employed to analyse TLR activity. KEY RESULTS: In MDDCs, AM3 induced or enhanced expression of CD54, CD83, CD86, HLA-DR, chemokines and chemokine receptors, interleukin (IL)-12p70 and IL-10. Furthermore, AM3 stimulated MDDCs to increase proliferation of allogenic T cells. AM3 triggered nuclear translocation of NF-kappaB and phosphorylation of p38 mitogen-activated protein kinase. AM3 promoted NF-kappaB activation in a TLR-4-dependent manner, and blocking TLR-4 activity attenuated the enhanced expression of CD80, CD83 and CD86 induced by AM3. AM3 enhanced the expression of maturation-associated markers in MDDCs from HCV-infected patients and increased the proliferation of T lymphocytes induced by these MDDCs. CONCLUSIONS AND IMPLICATIONS: These results underline the effects of AM3 in promoting maturation of MDDCs and suggest that AM3 might be useful in regulating immune responses in pathophysiological situations requiring DC maturation.

Identification of IgE binding polypeptides cross-reactive with the Parietaria judaica main allergenic polypeptide.

The major allergen of the pollen of Parietaria judaica was characterized using an anti-allergen MAb AC/15.1. The antibody was able to immunoadsorb four different polypeptides (10,000, 20,000, 30,000 and 40,000 mol. wt) from the pollen proteins radioiodinated by the Bolton-Hunter's reagent. The four polypeptides have been shown not to be covalently linked, except for the 10,000 mol. wt polypeptide (Pj10), which appeared to form Pj10 dimers under non-reducing conditions. All of them contained the antigenic epitope defined by the monoclonal antibody and demonstrated human IgE binding ability. The structural relationship of these polypeptides in the native allergen is discussed.

A competitive solid-phase radioimmunoassay for quantitation of the major allergen of Parietaria pollen.

A competitive solid-phase radioimmunoassay has been developed for quantitation of the major allergen of Parietaria judaica pollen. The assay is based on: (1) the ability of AC/1.1 monoclonal antibody to bind specifically to the P. judaica major allergen, and (2) the ability of crude pollen extracts or purified allergen to inhibit the binding of 125I-labelled allergen to solid-phase-bound AC/1.1 monoclonal antibody. The assay is sensitive enough to detect as little as 10 ng of allergen. A good correlation is found when the results obtained are compared with those produced by RAST inhibition (r = 0.95; P less than 0.001). Thus, this method can also be used for the estimation of the allergenic activity of P. judaica pollen extracts. The assay is easily completed in 2 h, allowing simultaneous analysis of a number of extracts.

Isolation of the major IgE-binding protein from Parietaria judaica pollen using monoclonal antibodies.

Allergen molecules from Parietaria judaica pollen, a widely distributed allergy inducer in Southern and Western Europe, have been studied using specific monoclonal antibodies (MAbs). MAbs against IgE-binding components were selected in a 4-step radioimmunoassay. Three different MAbs (AC/1.1, AC/7.1 and AC/15.1) were obtained which recognized epitope(s) located on a polypeptide of 10 Kd (Pj10). This polypeptide displayed the highest IgE-binding ability under either native or SDS-denatured conditions, as determined by immunoadsorption and immunodetection after SDS-PAGE, respectively. The Pj10-containing allergen, purified on an AC/1.1 MAb-Sepharose column, was able to inhibit most of the binding of specific IgE to the pollen extract coupled to paper discs in an inhibition radioallergosorbent test (RAST). The affinity-purified allergen exhibited the same immunoelectrophoretic behaviour as the native allergen.

Cross-reactivity between Parietaria judaica and Parietaria officinalis.

The relative allergenicities of Parietaria judaica and Parietaria officinalis have been studied by in vivo and in vitro techniques and a strong resemblance has been shown, with common allergenic polypeptides, though they differ in a group of anodic proteins. In vivo activity was very similar and both pollen extracts reacted with sera from patients who were sensitive to P. judaica, thus demonstrating a high rate of cross-reactivity. Extracts from both species may therefore be used interchangeably in diagnosis and immunotherapy.

Allergenic cross-reactivity among pollens of Urticaceae.

Common antigenic determinants have been observed between Parietaria and Urtica dioica pollen. The four Parietaria pollens selected (P. judaica, P. officinalis, P. lusitanica and P. mauritanica) are shown to possess a high allergenic homology. IgE-binding structures, homologous to the P. judaica main allergenic polypeptide (Pj10), were found in the other species by immunodetection. Monoclonal antibodies specific to the Pj10 polypeptide recognized proteins from the four Parietaria pollens. Skin prick test and RAST inhibition yielded results that also indicated a high allergenic cross-reactivity among these pollens, with homologous peptides bearing common antigenic and allergenic determinants. On the other hand, U. dioica pollen showed only a slight allergenic similarity to Parietaria. The potential allergenic activity of these pollens is discussed.

Distribution of allergen activity and identification of the main IgE-binding glycopeptide of Parietaria judaica pollen.

Parietaria judaica pollen extract was separated by preparative isoelectric focusing and showed two allergenic components (A1 and A2) heterogeneously distributed within the pH gradient (pH 3-10). By fused-rocket immunoelectrophoresis, A1 was detected from pH 3 to 10, whereas A2 was only found at pH values below 8. Both components, A1 and A2, showed IgE-binding ability throughout their pH ranges, as determined by crossed-radio immunoelectrophoresis. In addition, all the preparative isoelectric focusing fractions contained the main allergenic polypeptide of P. judaica (Pj10) when analyzed by SDS-PAGE. This polypeptide was shown to be a glycopeptide or to be bound to a glycoprotein with alpha-D-mannopyranoside or alpha-D-glycopyranoside residues. Implication of charged sugar residues in the wide distribution of P. judaica allergens in a pH gradient is discussed.

Identification and characterization of Parietaria judaica allergens.

Allergens of Parietaria judaica pollen extract have been identified and characterized biochemically. Two main allergenic components, A1 and A2, have been found by crossed-radioimmunoelectrophoresis and demonstrated to be spread in a wide range of pH. Immunoblotting studies revealed that at least eight SDS-denatured polypeptides show IgE-binding activity. The one exhibiting the highest allergenic activity, named Pj10 (MW 10,000 daltons) was found in all the fractions when the pollen extract was fractionated by chromatofocusing. Bidimensional electrophoretic analysis suggested that Pj10 either can form homopolymeric proteins of different molecular weights or can be associated to a number of proteins by disulfide bridges. Furthermore, Pj10 is the main molecular structure with IgE-binding activity in the two allergenic components A1 and A2 defined by immunological criteria.