Oral carrageenan induces antigen-dependent oral tolerance: prevention of anaphylaxis and induction of lymphocyte anergy in a murine model of food allergy

Immunosuppressive effects of carrageenan, a high-molecular-weight polysaccharide, on antibody and T cell responses have been previously demonstrated. However, its effect on anaphylaxis is unknown. Our objectives were to test carrageenan-mediated oral tolerance induction in young mice subsequently sensitized to a common cow's milk antigen. C3H/HeJ mice were fed or not lambda-carrageenan (0.5 g/L) and/or 0.01 mg/mL beta-lactoglobulin (BLG) for 5 d before oral sensitization with BLG and cholera toxin. Subsequently, the mice were challenged with BLG and symptom scores of anaphylaxis were recorded. Mesenteric lymph node cells, spleen cells, Peyer's patches cells, intraepithelial lymphocytes, and lamina propria lymphocytes were isolated and stimulated in vitro with BLG, IL-2, or left unstimulated. BLG-specific IgG, IgG(1), and IgG(2a) antibodies were measured. Pretreatment with carrageenan and BLG, but not pretreatment with either carrageenan or BLG alone or omission of pretreatment, diminished significantly the number of anaphylactic mice after BLG challenge (6.3 % versus 53 % in mice without pretreatment, p = 0.006). Mesenteric lymph nodes and spleen cells from pretreated mice proliferated less in presence of BLG or IL-2 than cells from sensitized control mice. Antigen-specific antibody production and passive cutaneous anaphylaxis was not suppressed by carrageenan and BLG pretreatment. In conclusion, carrageenan administered to young mice in conjunction with low doses of allergen before sensitization efficiently prevents anaphylaxis

Multidrug resistance in tumour cells: characterisation of the multidrug resistant cell line K562-Lucena 1

Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.<br>A resistência a múltiplos fármacos é o principal obstáculo no tratamento de pacientes com câncer. O mecanismo responsável pela resistência múltipla mais bem caracterizado envolve a expressão do produto do gene MDR-1, a glicoproteína P. Entretanto, o processo de resistência tem fatores múltiplos. Estudos de mecanismos de resistência m��ltipla a fármacos têm dependido da análise de linhagens celulares tumorais que foram selecionadas e apresentam reatividade cruzada a uma ampla faixa de agentes anti-tumorais. Este trabalho caracteriza uma linhagem celular com múltipla resistência a fármacos, selecionada originalmente pela resistência ao alcalóide de Vinca vincristina e derivado da linhagem eritro-leucêmica K562. Esta linhagem celular, denominada Lucena 1, super-expressa a glicoproteína P e tem sua resistência revertida pelos quimio-sensibilizantes verapamil, trifluoperazina e ciclosporinas A, D e G. Ademais, demonstramos que o azul de metileno era capaz de reverter parcialmente a resistência nesta linhagem celular. Em contraste, o uso de 5-flúor-uracil aumentava a resistência de Lucena 1. Adicionalmente aos quimioterápicos, células Lucena 1 eram resistentes radiação ultra-violeta A e peróxido de hidrogênio e deixavam de mobilizar o cálcio intra-celular quando se usava tapsigargina. Mudanças no cito-esqueleto desta linhagem foram também observadas

Wnt/β-catenin pathway regulates <it>ABCB1</it> transcription in chronic myeloid leukemia

<p>Abstract</p> <p>Background</p> <p>The advanced phases of chronic myeloid leukemia (CML) are known to be more resistant to therapy. This resistance has been associated with the overexpression of <it>ABCB1</it>, which gives rise to the multidrug resistance (MDR) phenomenon. MDR is characterized by resistance to nonrelated drugs, and P-glycoprotein (encoded by <it>ABCB1</it>) has been implicated as the major cause of its emergence. Wnt signaling has been demonstrated to be important in several aspects of CML. Recently, Wnt signaling was linked to <it>ABCB1</it> regulation through its canonical pathway, which is mediated by β-catenin, in other types of cancer. In this study, we investigated the involvement of the Wnt/β-catenin pathway in the regulation of <it>ABCB1</it> transcription in CML, as the basal promoter of <it>ABCB1</it> has several β-catenin binding sites. β-catenin is the mediator of canonical Wnt signaling, which is important for CML progression.</p> <p>Methods</p> <p>In this work we used the K562 cell line and its derived MDR-resistant cell line Lucena (K562/VCR) as CML study models. Real time PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), flow cytometry (FACS), western blot, immunofluorescence, RNA knockdown (siRNA) and Luciferase reporter approaches were used.</p> <p>Results</p> <p>β-catenin was present in the protein complex on the basal promoter of <it>ABCB1</it> in both cell lines <it>in vitro</it>, but its binding was more pronounced in the resistant cell line <it>in vivo</it>. Lucena cells also exhibited higher β-catenin levels compared to its parental cell line. <it>Wnt1</it> and <it>β-catenin</it> depletion and overexpression of nuclear β-catenin, together with TCF binding sites activation demonstrated that <it>ABCB1</it> is positively regulated by the canonical pathway of Wnt signaling.</p> <p>Conclusions</p> <p>These results suggest, for the first time, that the Wnt/β-catenin pathway regulates <it>ABCB1</it> in CML.</p