Growth modulation of human cells in vitro by mild oxidative stress and 1,4-dihydropyridine derivative antioxidants.

Reactive oxygen species and lipid peroxidation products are not only cytotoxic but may also modulate signal transduction in cells. Accordingly, antioxidants may be considered as modifiers of cellular redox signaling. Therefore, the effects of two novel synthetic antioxidants, analogues of 1,4-dihydropyridine derivatives, cerebrocrast and Z41-74 were analysed in vitro on human osteosarcoma cell line HOS, the growth of which can be modulated by lipid peroxidation. The cells were pretreated with either cerebrocrast or Z41-74 and afterwards exposed to mild, copper induced lipid peroxidation or to 4-hydroxynonenal (HNE), the end product of lipid peroxidation. The results obtained have shown that both antioxidants exert growth modulating effects interfering with the lipid peroxidation. Namely, cells treated with antioxidants showed increased metabolic rate and cell growth, thereby attenuating the effects of lipid peroxidation. Such biomodulating effects of cerebrocrast and Z41-74 resembled growth modulating effects of HNE, suggesting that the antioxidants could eventually promote cellular adaptation to oxidative stress interacting with redox signaling and hydroxynonenal HNE-signal transduction pathways. This may be of particular relevance for better understanding the beneficial role of hydroxynonenal HNE in cell growth control. Therefore, cerebrocrast and Z41-74 could be convenient to study further oxidative homeostasis involving lipid peroxidation.

Reversal of multidrug resistance in murine lymphoma cells by amphiphilic dihydropyridine antioxidant derivative.

BACKGROUND: Multidrug resistance, the principal mechanism by which cancer cells develop resistance to chemotherapy drugs, is a major factor in the failure of many forms of chemotherapies. AIM: The aim of the study was to investigate the effect of K-2-11 on the reversal of multidrug resistance. MATERIALS AND METHODS: The effects of amphiphilic dihydropyridine derivative K-2-11 were tested on MDR1-expressing mouse lymphoma cells and their parental control. The effects of K-2-11 with and without doxorubicin were studied by determination of cell viability, cell proliferation and production of reactive oxygen species. RESULTS: K-2-11 caused complete reversal of multidrug resistance of the MDR cells, being much more efficient than the positive control verapamil. Accordingly, the cytotoxic effects of doxorubicin were enhanced by K-2-11, both in the MDR and in parental cell line, while K-2-11 alone did not affect cell viability. K-2-11 also acted as an antioxidant, reducing the cellular generation of reactive oxygen species. CONCLUSION: Our results indicate the high potential of K-2-11 as a novel antioxidant with potent MDR-blocking ability that should be studied further for development in adjuvant anticancer treatments.

Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1.

Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1-6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.

Antigen presentation of the immunodominant T-cell epitope of the major mugwort pollen allergen, Art v 1, is associated with the expression of HLA-DRB1 *01.

BACKGROUND: Mugwort pollen allergens are the main cause of pollinosis in late summer in Europe. Ninety-five percent of patients allergic to mugwort are sensitized to the major allergen Art v 1. In contrast to other common pollen allergens that contain multiple T-cell epitopes, Art v 1 contains only 1 immunodominant T-cell epitope (Art v 1 25-36 ). OBJECTIVE: To characterize the minimal epitope of Art v 1 25-36 and to investigate a possible association of Art v 1 reactivity with HLA class II phenotypes. METHODS: Art v 1-specific T-cell lines and clones were established from 51 patients with clinically defined mugwort pollen allergy and IgE specific for Art v 1. To define minimal epitopes and binding sites within Art v 1 25-36 , truncated and single-substitution analog peptides were used for T-cell stimulation. To study HLA restriction, monoclonal anti-HLA antibodies and antigen-presenting cells with defined HLA-DRB and -DQB1 alleles were used. HLA typing of patients with allergy was performed by hybridization with sequence-specific oligonucleotides, PCR, and nucleotide sequencing. RESULTS: In 96% of the patients, a cellular response to Art v 1 25-36 was obtained, and a core region of 5 to 10 amino acids containing 3 to 5 amino acids essential for T-cell reactivity was defined. The frequency of HLA-DRB1 * 01 in patients recognizing Art v 1 25-36 was significantly increased as compared with healthy controls (69% vs 21%; odds ratio, 8.45; P < 10 -6 ), and HLA-DRB1 * 01 was identified as the main restriction element for the presentation of the immunodominant epitope. CONCLUSIONS: Allergy to Art v 1 is characterized by a uniform T-cell response. The disease is apparently associated with the HLA-DR1 phenotype. Therefore, mugwort pollinosis is an ideal candidate for a peptide-based immunotherapy.

Biology of weed pollen allergens.

Weeds represent a heterogeneous group of plants, usually defined by no commercial or aesthetic value. Important allergenic weeds belong to the plant families Asteraceae, Amaranthaceae, Urticaceae, Euphorbiaceae, and Plantaginaceae. Major allergens from ragweed, mugwort, feverfew, pellitory, goosefoot, Russian thistle, plantain, and Mercurialis pollen have been characterized to varying degrees. Four major families of proteins seem to be the major cause of allergic reactions to weed pollen: the ragweed Amb a 1 family of pectate lyases; the defensin-like Art v 1 family from mugwort, feverfew, and probably also from sunflower; the Ole e 1-like allergens Pla l 1 from plantain and Che a 1 from goosefoot; and the nonspecific lipid transfer proteins Par j 1 and Par j 2 from pellitory. As described for other pollens, weed pollen also contains the panallergens profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients.

Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain.

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.