Cholera toxin (CT) and heat-labile enterotoxin (LT) are powerful mucosal adjuvants whose cellular targets and mechanism of action are unknown. There is emerging evidence that dendritic cells (DC) are one of the principal cell types that mediate the adjuvant effects of these toxins in vivo. Here we investigate the effects of CT and LT on the maturation of human monocyte-derived DC (MDDC) in vitro. We found that an enzymatically active A domain is necessary for both CT and LT to induce the maturation of MDDC and that this activation is strictly cyclic AMP (cAMP) dependent. ADP-ribosylation-defective derivatives of these toxins failed to induce maturation of MDDC, whereas dibutyryl-cyclic-3,5-AMP and Forskolin mimic the maturation of MDDC induced by CT and LT. In addition, an inhibitor of cAMP-dependent kinases, Rp-8-Br-cAMPs, blocked the ability of CT, LT, and Forskolin to activate MDDC. CT, LT, dibutyryl-cyclic-3,5-AMP, and Forskolin also dominantly inhibit interleukin 12 and tumor necrosis factor alpha production by MDDC in the presence of saturating concentrations of lipopolysaccharide. Taken together, these results show that the effects of CT and LT on MDDC are mediated by cAMP.Cholera toxin (CT) and heat-labile enterotoxin (LT) are AB5 enterotoxins produced by Vibrio cholerae and enteropathic Escherichia coli, the primary causative agents of cholera and traveler's diarrhea, respectively. CT and LT consist of a 27-kDa catalytic A domain anchored in a ring of five identical, 11.7-kDa B subunits (18). The B pentamers of these toxins bind to gangliosides on cell membranes (12). The B pentamer of CT (CTB) binds exclusively to GM1 gangliosides, while the B pentamer of LT (LTB) binds to other gangliosides in addition to GM1 (9). These toxins exploit the host protein retention and degradation pathways to gain access to the cytoplasm, reviewed in reference 11. In the cytosol, their A1 subunits catalyze the transfer of an ADP-ribose from NAD to stimulatory ␣-subunits of G proteins (Gs␣). After ADP-ribosylation, Gs␣ binds to adenylate cyclase and constitutively activates it, leading to a sustained increase in intracellular cyclic AMP (cAMP) concentration (4).CT and LT are also powerful mucosal immunogens and adjuvants, reviewed in reference 14. In mice, antibody responses to CT and bystander antigens can last up to 2 years (15, 26). CT has been shown to induce primarily Th2 responses, characterized by CD4 ϩ T cells producing interleukin 4 (IL-4), IL-5, IL-6, and IL-10 and by the production of immunoglobulin G1 (IgG1), IgA, and IgE antibodies (17,29). By contrast, LT has been reported to induce mixed Th1 and Th2 responses (24). It has been proposed that differences in the ganglioside binding specificities of their B pentamers contribute to their discordant Th1/Th2 patterns (30, 31). Although these toxins are extensively used as adjuvants in animal models, their toxicity makes them unsuitable for human use. For this reason, a number of investigators have attempted to identify nontoxic derivatives of CT and LT that retain ...

The infection of CD4؉ host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.

Astrocytoma is the most common malignant brain tumor in humans. Loss of the p53 signaling pathway and up-regulation of the ras signaling pathway are common during tumor progression. We have shown previously that mice mutant for Trp53 and Nf1 develop astrocytoma, progressing to glioblastoma, on a C57BL͞6J strain background. In contrast, here we present data that mice mutant for Trp53 and Nf1 on a 129S4͞SvJae background are highly resistant to developing astrocytoma. Through analysis of F 1 progeny, we demonstrate that susceptibility to astrocytoma is linked to chromosome 11, and that the modifier gene(s) responsible for differences in susceptibility is closely linked to Nf1 and Trp53. Furthermore, this modifier of astrocytoma susceptibility is itself epigenetically modified. These data demonstrate that epigenetic effects can have a strong effect on whether cancer develops in the context of mutant ras signaling and mutant p53, and that this mouse model of astrocytoma can be used to identify modifier phenotypes with complex inheritance patterns that would be unidentifiable in humans. Because analysis of gene function in the mouse is often performed on a mixed C57BL͞6,129 strain background, these data also provide a powerful example of the potential of these strains to mask interesting gene functions.A strocytoma is a characteristically diffuse tumor of the central nervous system (CNS). Because of its diffuse infiltration, it often cannot be completely resected, leading to a very poor prognosis for patients. Astrocytoma, together with glioblastoma (the highest grade of astrocytoma), accounts for more than three-quarters of all gliomas, making it the most common malignant brain tumor (1). The 5-year survival rate for glioblastoma is Ͻ3%. A better understanding of the genetic risk factors associated with astrocytoma will give insight into the mechanism of astrocytoma initiation and progression and will lead to better screening methods and new targets for therapy.Data from human populations pointing to genetic risk factors for astrocytoma are sparse. Malmer et al. (2) have examined the increased family risk of developing low-vs. high-grade glioma and favor the view that autosomal recessive genes affect astrocytoma risk, although the role of a common environment in familial risk cannot be excluded. Several familial cancer syndromes show an increased risk for astrocytoma, including neurofibromatosis type 1 (NF1) (3, 4) and Li-Fraumeni syndrome (LFS) (5). NF1 patients have a mutation in the NF1 gene (6) (Nf1 in the mouse) and are predisposed to neurofibromas and optic gliomas, with an increased risk for malignant peripheral nerve sheath tumors and diffuse astrocytoma͞glioblastoma (3, 4). Studies of NF1 families have demonstrated a role for modifier genes unlinked to NF1 in the severity of the disease with respect to the numbers of neurofibromas and the presence or absence of optic gliomas (7). Studies of NF1 patients have also shown that patients with optic glioma are more likely to develop CNS tumors such as astrocytoma ...