Background and purpose: Allergic inflammation and autoimmune diseases, such as atopic dermatitis, psoriasis and multiple sclerosis (MS), involve both mast cell and T‐cell activation. However, possible interactions between the two and the mechanism of such activations are largely unknown. Experimental approach: Human umbilical cord blood‐derived cultured mast cells (hCBMCs) and Jurkat T cells were incubated separately or together, following activation with myelin basic protein (MBP), as well as with or without pretreatment with the flavonoid luteolin for 15 min. The supernatant fluid was assayed for inflammatory mediators released from mast cells and interleukin (IL)‐2 release from Jurkat cells. Key results: MBP (10 μM) stimulates hCBMCs to release IL‐6, IL‐8, transforming growth factor (TGF)‐β1, tumour necrosis factor‐α (TNF‐α), vascular endothelial growth factor (VEGF), histamine and tryptase (n=6, P<0.05). Addition of mast cells to Jurkat cells activated by anti‐CD3/anti‐CD28 increases IL‐2 release by 30‐fold (n=3, P<0.05). MBP‐stimulated mast cells and their supernatant fluid further increase Jurkat cell IL‐2 release (n=3, P<0.05). Separation of mast cells and activated Jurkat cells by a Transwell permeable membrane inhibits Jurkat cell stimulation by 60%. Pretreatment of Jurkat cells with a TNF‐neutralizing antibody reduces IL‐2 release by another 40%. Luteolin pretreatment inhibits mast cell activation (n=3–6, P<0.05), Jurkat cell activation and mast cell‐dependent Jurkat cell stimulation (n=3, P<0.05). Conclusions and implications: Mast cells can stimulate activated Jurkat cells. This interaction is inhibited by luteolin, suggesting that this flavonoid may be useful in the treatment of autoimmune diseases. British Journal of Pharmacology (2008) 155, 1076–1084; doi:; published online 22 September 2008

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes methods for culturing Ae. aegypti and includes a procedure for egg collection that can be used in conjunction with fixation, immunohistochemistry, and in situ protocols.