Activated mast cells reside in close apposition to T cells in some inflammatory processes. In this study, we analyzed whether this close physical proximity affects human mast cell degranulation and cytokine release. Thus HMC-1 human mast cells or primary bone marrow-derived human mast cells were cocultured with activated and with resting T cells. Mast cells cocultured with activated T cells released histamine and ␤-hexosaminidase and produced tumor necrosis factor ␣ (TNF-␣), an effect that peaked at 20 h. Kinetics of histamine release paralleled the formation of heterotypic aggregates. Separation of the two cell populations with a porous membrane prevented mediator release and TNF-␣ production. Addition of the PI3-kinase inhibitor, wortmannin, inhibited the heterotypic adhesion-associated degranulation but not TNF-␣ production. These data thus indicate a novel pathway through which human mast cells are activated to both release granule-associated mediators and to produce cytokines in association with heterotypic adhesion to activated human T cells.

The cytoplasmic tails of both the ␤ and ␥ subunits of the high affinity IgE receptor (Fc⑀RI) contain a consensus sequence termed the immunoreceptor tyrosinebased activation motif (ITAM). This motif plays a critical role in receptor-mediated signal transduction. Synthetic peptides based on the ITAM sequences of the ␤ and ␥ subunits of Fc⑀RI were used to investigate which proteins associate with these motifs. Tyrosine-phosphorylated ␤ and ␥ ITAM peptides immobilized on beads precipitated Syk, Lyn, Shc, Grb2, and phospholipase C-␥1 from lysates of rat basophilic leukemia RBL-2H3 cells. Syk was precipitated predominantly by the tyrosine-diphosphorylated ␥ ITAM peptide, but much less by the diphosphorylated ␤ ITAM peptide or by the monophosphorylated peptides. Phospholipase C-␥1, Shc, and Grb2 were precipitated only by the diphosphorylated ␤ ITAM peptide. Non-phosphorylated ITAM peptides did not precipitate these proteins. In membrane binding assays, fusion proteins containing the Src homology 2 domains of phospholipase C-␥1, Shc, Syk, and Lyn directly bound the tyrosine-phosphorylated ITAM peptides. Although the ITAM sequences of the ␤ and ␥ subunits of Fc⑀RI are similar, once they are tyrosinephosphorylated they preferentially bind different downstream signaling molecules. Tyrosine phosphorylation of the ITAM of the ␥ subunit recruits and activates Syk, whereas the ␤ subunit may be important for the Ras signaling pathway.Aggregation of the high affinity IgE receptors (Fc⑀RI) 1 on basophils and mast cells initiates a cascade of events that results in the release of inflammatory mediators. This pathway includes the activation of several protein-tyrosine kinases such as Lyn, Syk, Btk, and Fak that induce the tyrosine phosphorylation of various proteins (1-3). There is also stimulation of phospholipase A 2 , C, and D, the mobilization of Ca 2ϩ from intracellular and extracellular sources and activation of serine and threonine kinases (4, 5).The high affinity IgE receptor on mast cells and basophils is a tetrameric structure composed of the IgE binding ␣ chain, a ␤ subunit, and a homodimer of disulfide-linked ␥ chains (6). The COOH-terminal cytoplasmic domain of Fc⑀RI␤ and the cytoplasmic domain of Fc⑀RI␥ appear to be important in receptor-mediated signal transduction (7,8). These transducing subunits contain a cytoplasmic motif with the amino acid sequence (D/E)X 2 YX 2 LX 6 -7 YX 2 (L/I) that is critical for cell activation (9 -11). This motif called ITAM (for Immunoreceptor T yrosinebased Activation Motif) (12) is present in the ␤ and ␥ subunits of Fc⑀RI, in the subunit of the T-cell receptor complex, and in Ig␣ and Ig␤ of the B-cell receptor. This motif contains all the structural information essential for signal transduction and is rapidly tyrosine-phosphorylated after receptor aggregation (11,(13)(14)(15). Phosphorylation of the tyrosine residues appears to be necessary as mutants where the tyrosines are replaced with phenylalanine are inactive (16,17). This phosphorylation is probably due to src family tyrosine k...

No abstract

Bacillus anthracis, the etiological agent of anthrax disease, is a proven weapon of bioterrorism. Currently, the only licensed vaccine against anthrax in the United States is AVA Biothrax, which, although efficacious, suffers from several limitations. This vaccine requires six injectable doses over 18 months to stimulate protective immunity, requires a cold chain for storage, and in many cases has been associated with adverse effects. In this study, we modified the B. anthracis protective antigen (PA) gene for optimal expression and stability, linked it to an inducible promoter for maximal expression in the host, and fused it to the secretion signal of the Escherichia coli alpha-hemolysin protein (HlyA) on a low-copy-number plasmid. This plasmid was introduced into the licensed typhoid vaccine strain, Salmonella enterica serovar Typhi strain Ty21a, and was found to be genetically stable. Immunization of mice with three vaccine doses elicited a strong PA-specific serum immunoglobulin G response with a geometric mean titer of 30,000 (range, 5,800 to 157,000) and lethal-toxinneutralizing titers greater than 16,000. Vaccinated mice demonstrated 100% protection against a lethal intranasal challenge with aerosolized spores of B. anthracis 7702. The ultimate goal is a temperature-stable, safe, oral human vaccine against anthrax infection that can be self-administered in a few doses over a short period of time.