SUMMARYAntibodies recognizing anionic phosphoiipids have bren described in systemic iupus erythemalosus (SLE) and other autoimmune diseases. Recent studies have shown that some of these antibodies may recognize a cardiolipin-binding protein (apolipoprotein H) rather than phosphoiipids. A similar possibility is conceivable for other cardiolipin-binding proieins that are targets of auioaniibodies. In this study we have addressed whether this might be the case for histones, a set of highly cationic and widely distributed proteins that react in a well known auloantibody system. Our results indicate that: (i) histones bind to anionic phosphoiipids (cardioiipin and phosphatidylserine) with high avidity, but not to zwitterionic phosphoiipids (phosphatidylcholine); (ii) monoclonal and polyclona! antihistone antibodies recognize histones bound to cardioiipin; (iii) the addition of histones to serum samples containing antihistone antibodies often enhances their anticardiolipin reactivity. In addition, we have found that antihistone-producing hybridomas derived from MKL-tpr mice may show anticardiolipin activity due to the presence of histones in the cell culture supernatants with the resultant formation of immune complexes. Taken together, the results suggest a potential role for histones in the anticardiolipin activity detected in sera containing antihistone antibodies. These histone phospholipid interactions should be taken into account when evaluating the pathogenic effects of antihistone antibodies or other autoantibodies reacting with nuclear components (e.g. nucleosomes) containing histones.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

We report that 100% mice survival after tumor challenge is achieved with cytokine-engineered cells employing nonviral lipoplexes and without using viral vectors. We describe this effect with cytokine-secreting tumor cell vaccines, based on cell clones or fresh transfected cells. Tumor cells were transfected with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-4 plasmids employing the cationic lipid DOTAP, were irradiated (150 Gy) and kept frozen until use. The transfection efficacy was analyzed by qRT-PCR and flow cytometry. Vaccination induced potent antitumor rejection, resulting in 100% mice survival. Furthermore, the antitumor immunity was long lasting, since a two-fold survival delay was observed in mice after tumor rechallenge (6 months later). While cell clones secreting GM-CSF were the most effective in wild-type tumor cell rejection, little or no effect was observed with clones secreting IL-4. We found similar antitumor efficacy employing fresh transfected cells by nonviral procedures, demonstrating that cells genetically modified by nonviral vectors (both clones and fresh transfected cells) are a safe and efficient tool for antitumor vaccines. These vaccines allow us to achieve the highest antitumor efficacy based on nonviral gene therapy techniques. In addition, the vaccination success with fresh transfected cells simplifies the procedure and provides new insights into the clinical application of nonviral gene therapy procedures.

Here we studied and characterized different peripheral blood (PB) regulatory T cell (Treg) subsets in rheumatoid arthritis (RA) patients and tested the hypothesis that changes in these cells can be linked to the degree of inflammation and relapsing/remission periods. PB cells were examined from RA subjects (n = 60) with different disease activity score-28 (DAS28) and from healthy controls (n = 40). Frequencies of Treg subsets expressing characteristic membrane antigens, FoxP3 or intracellular cytokines were quantified by flow cytometry. We observed a decrease in the percentages of CD4(+)CD25(high), CD4(+)CD25(int), CD4(+)CD25(int/high)FoxP3(+), CD4(+)CD38(+), CD4(+)CD62L(+), CD8(+)CD25(high)CD45RA(+) and CD8(+)CD25(int)CD45RA(+) T cells in PB of RA patients compared to healthy controls. In addition, we found increased percentages of cells expressing membrane/intracellular regulatory antigens such as OX40 (CD134), CD45RB(low) or CTLA-4 (CD152), and a higher proportion of other T cell subsets including CD4(+)CTLA-4(+), CD4(+)IL10(+), CD4(+)CD25(int)IL10(+), CD4(+)CD25(int) TGFbeta(+), CD4(+)CD25(low) TGFbeta(+) and CD8(+)CD28(- ). We show that most of these changes parallel the intensity of inflammation, with lowest or highest values in patients with moderately/very active disease compared to healthy controls and at times to patients with inactive RA. The balance between these cell subsets and their antigen expression would determine the inflammation levels and could thus be linked to the relapsing/remission periods of the disease.

Hypersensitivity to carmine (E120) has been identified as a cause of food intolerance and occupational asthma. We present a case of occupational asthma following exposure to carmine in a manufacturer of sausages and review the literature. Case Report: A 42-year-old non-atopic male presented with a 5-year history of rhinoconjunctivitis and asthma on occupational exposure to food additive dusts. Symptoms increased after work. The patient had been exposed for more than 20 years. Methods: Skin prick tests were performed with a battery of common inhalant allergens and spices. Cochineal, carmine lake and additive mixes used by the patient were extracted and subsequently used for skin prick test, bronchial provocation and in vitro measurements (specific IgE, Western blot and chromatographic fractionation). Results: Prick tests were positive to carmine and carmine-containing additives; carmine-specific IgE and bronchial challenge tests were also positive (PC₂₀ = 0.0004 mg/ml and 1.6 kU/l). Western blot showed IgE binding to bands of about 30 kDa on cochineal extract and a diffuse pattern at 40–97 kDa on carmine. This result was confirmed by gel filtration chromatography and dot blot. Carmine completely inhibited IgE binding to cochineal extract. Discussion: Carmine is a potential sensitizer in an occupational setting: 18 cases of occupational asthma have been described to date. Carmine allergens are poorly defined; in general, proteins from cochineal not removed by the extraction process are considered as the main allergens in carmine. Our results are consistent with this, but show that these proteins may be subject to chemical modification.