The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow-derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of Fc(epsilon)RI, Syk, and Vav was intact in LAT-deficient BMMCs following Fc(epsilon)RI engagement, tyrosine phosphorylation of SLP-76, PLC-gamma1, and PLC-gamma2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after Fc(epsilon)RI cross-linking. These results show that LAT plays a critical role in Fc(epsilon)RI-mediated signaling in mast cells.

The hematopoietic cell-specific protein Vav1 is a substrate of tyrosine kinases activated following engagement of many receptors, including FcRI. Vav1-deficient mice contain normal numbers of mast cells but respond more weakly than their normal counterparts to a passive systemic anaphylaxis challenge. Vav1-deficient bone marrow-derived mast cells also exhibited reduced degranulation and cytokine production, although tyrosine phosphorylation of FcRI, Syk, and LAT (linker for activation of T cells) was normal. In contrast, tyrosine phosphorylation of phospholipase C␥1 (PLC␥1) and PLC␥2 and calcium mobilization were markedly inhibited. Reconstitution of deficient mast cells with Vav1 restored normal tyrosine phosphorylation of PLC␥1 and PLC␥2 and calcium responses. Thus, Vav1 is essential to FcRI-mediated activation of PLC␥ and calcium mobilization in mast cells. In addition to its known role as an activator of Rac1 GTPases, these findings demonstrate a novel function for Vav1 as a regulator of PLC␥-activated calcium signals.The early events following activation of the high-affinity receptor for immunoglobulin E (IgE) (FcεRI) on mast cells are well studied (32). Like other immunoreceptors, FcεRI contains multiple subunits, the IgE binding ␣ chain, and the ␤ and ␥ chains that function to transduce signals via the paired tyrosine residues of the immunoreceptor tyrosine-based activation motifs (ITAMs) that are found within (45). Aggregation of multiple IgE-occupied FcεRI by polyvalent antigen (Ag) leads to transphosphorylation of the ␤-and ␥-chain ITAMs by the associated Src family protein tyrosine kinase (PTK) Lyn (17,41). This creates a new binding surface for Syk PTK, whose tandem Src homology 2 (SH2) domains facilitate the interaction with the ITAM, resulting in activation of this enzyme (4, 31). Activated Syk then phosphorylates multiple substrates, among which the linker for activation of T cells (LAT) is proximal and essential to FcεRI-activated responses (48). LAT, the SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), and phospholipase C␥ (PLC␥) have all been implicated in the regulation of calcium responses in mast cells (40,48,59) and in other cells (12,59,64). These proteins, along with the Rac GTPase-activating Vav1 protein, interact to form a functional macromolecular complex at the plasma membrane that regulates both Rac and Ras signaling (1,14,21,37,51).Vav1 is a cytosolic protein primarily expressed in hematopoietic cells whose structure and function have been extensively examined (6). Its structure includes a calponin homology (CH) domain, a Dbl homology (DH) domain, a pleckstrin homology (PH) domain, a single SH2 domain, and two Src homology 3 (SH3) domains that flank the SH2 domain. Functional studies demonstrated that tyrosine phosphorylation activates Vav's guanine nucleotide exchange factor (GEF) activity for Rho family GTPases (15) with a clear preference for Rac GTPases. Vav1 also promotes intracellular signaling by its C-terminal Grb2-like adapter region, which contains an SH2...

These studies were conducted to investigate the potential use of a flow cytometric analysis method for the identification and differentiation of chemicals with the capacity to induce irritation, IgE- or T cell-mediated hypersensitivity responses. An initial study investigated the ability of equally sensitizing concentrations (determined by local lymph node assay) of IgE-mediated (Toluene Diisocyanate-TDI) and T cell-mediated (Dinitrofluorobenzene-DNFB) allergens to differentially modulate the IgE+B220+ population in the lymph nodes draining the dermal exposure site. Sodium lauryl sulfate (SLS) was also tested as a nonsensitizing irritant control. Female B6C3F1 mice were dermally exposed once daily for 4 consecutive days, with the optimum time point for analysis determined by examining the IgE+B220+ population 8, 10, and 12 days post-initial chemical exposure. At the peak time point, day 10, the IgE+B220+ population was significantly elevated in TDI (41%), while moderately elevated in DNFB (18%) exposed animals when compared to the vehicle (0.8%), and remained unchanged in SLS (2.2%) exposed animals when compared to the ethanol control (2.5%). Experiments in our laboratory and others have demonstrated that the draining lymph node B220+ population becomes significantly elevated following exposure to allergens (IgE- and T cell-mediated), not irritants, allowing for their differentiation. An existing mouse ear swelling assay was used to identify chemical irritants. Therefore, using the endpoints of percent ear swelling, percent B220+ cells, and percent IgE+B220+ cells, a combined irritancy/phenotypic analysis assay was developed and tested with tetradecane (irritant), toluene diisocyanate, trimellitic anhydride (IgE-mediated allergens), benzalkonium chloride, dinitrofluorobenzene, oxazolone, and dinitrochlorobenzene (T cell-mediated allergens) over a range of concentrations. Based upon the pattern of response observed, a paradigm was developed for continued evaluation: Irritant exposure will result in significant ear swelling without altering the B220+ or IgE+B220+ populations. Exposure to sensitizers (IgE-mediated or T cell-mediated) will increase the B220+ population and the percent ear swelling will remain unchanged or will significantly increase, depending on the irritancy capacity of the chemical. Both the IgE+B220+ and B220+ populations will become elevated at the same test concentration following exposure to IgE-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will be equal to the percent of B220+ cells. The B220+ population will increase at a lower test concentration than the IgE+B220+ population, following exposure to T cell-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will reach less than half that of the percent of B220+ cells. The irritancy/phenotypic analysis method may represent a single murine assay able to identify and differentiate chemicals with the capacity to induce irritation, or IgE-mediated or T cell-mediate...

The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies have been performed in mice or rabbits to determine where in the body these plasmid vaccines would biodistribute and how rapidly they would clear. In the course of these studies, it has been observed that regardless of the gene insert (expressing the vaccine immunogen or cytokine adjuvant) and regardless of the promoter used to drive expression of the gene insert in the plasmid backbone, the plasmid vaccines do not biodistribute widely and remain essentially in the site of injection, in the muscle and overlying subcutis. Even though approximately 10(14) molecules are inoculated in the studies in rabbits, by day 8 or 9 ( approximately 1 week postinoculation), already all but on the order of 10(4)-10(6) molecules per microgram of DNA extracted from tissue have been cleared at the injection site. Over the course of 2 months, the plasmid clears from the site of injection with only a small percentage of animals (generally 10-20%) retaining a small number of copies (generally around 100 copies) in the muscle at the injection site. This pattern of biodistribution (confined to the injection site) and clearance (within 2 months) is consistent regardless of differences in the promoter in the plasmid backbone or differences in the gene insert being expressed by the plasmid vaccine. In addition, integration has not been observed with plasmid vaccine candidates inoculated i.m. by Biojector 2000 or by needle and syringe. These data build on the repeated-dose toxicology studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.