Induction of germline (GL) epsilon transcripts, an essential step preceding Ig isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g., CD40 ligand or LPS. We demonstrate that AP-1 (Fos and Jun), induced transiently by CD40 ligand or LPS, binds a DNA element in the mouse GL epsilon promoter. AP-1 synergizes with Stat6 to activate both the intact GL epsilon promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL epsilon promoter. By contrast, C/EBP beta, which trans-activates the human GL epsilon promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not trans-activate the human GL epsilon promoter. Thus, induction of GL epsilon transcripts in mice and humans may be regulated differently. In addition, although mouse GL epsilon transcripts have a half-life of approximately 100 min, the RNA level continues to increase for up to 24 h, and the promoter appears to be active for at least 2 days after B cell activation. Altogether, these data suggest that induction of AP-1 activity, although transient, is required for activation of the mouse GL epsilon promoter by IL-4-induced Stat6.

Anti-Gal is the most abundant antibody in humans, constituting 1% of immunoglobulins. Anti-Gal binds specifically alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). Immunogenicity of autologous tumor associated antigens (TAA) is greatly increased by manipulating tumor cells to express alpha-gal epitopes and bind anti-Gal. Glycolipids with alphagal epitopes (alpha-gal glycolipids) injected into tumors insert into the tumor cell membrane. Anti-Gal binding to the multiple alpha-gal epitopes de novo presented on the tumor cells results in targeting of these cells to APC via the interaction between the Fc portion of the bound anti-Gal and Fcgamma; receptors on APC. The APC process and present immunogenic TAA peptides and thus, effectively activate tumor specific CD4+ helper T cells and CD8+ cytotoxic T cells which destroy tumor cells in micrometastases. The induced immune response is potent enough to overcome immunosuppression by Treg cells. A phase I clinical trial indicated that alpha-gal glycolipid treatment has no adverse effects. In addition to achieving destruction of micrometastases in cancer patients with advance disease, alpha-gal glycolipid treatment may be effective as neo-adjuvant immunotherapy. Injection of alpha-gal glycolipids into primary tumors few weeks prior to resection can induce a protective immune response capable of destroying micrometastases expressing autologous TAA, long after primary tumor resection.

Betafectin PGG-Glucan, a novel beta-(1,6) branched beta-(1,3) glucan purified from the cell walls of Saccharomyces cerevisiae, has been shown to synergize with myeloid growth factors in vitro and to enhance hematopoietic recovery in myelosuppressed mice and primates. Here we report that PGG-Glucan is also capable of mobilizing peripheral blood progenitor cells (PBPC). PGG-Glucan (0.5 mg/kg to 16 mg/kg) was administered intravenously to C3H/HeN male mice and blood collected at times ranging from 30 min to seven days after injection. Based on granulocyte-macrophage colony-forming cell (GM-CFC) levels, peak mobilization occurred 30 min after a 2 mg/kg PGG-Glucan dose. At this time GM-CFC numbers in PGG-Glucan-treated mice were approximately fourfold greater than in saline-treated control mice. A second, smaller wave of GM-CFC mobilization (approximately twofold increase) also occurred on days 4 and 5 after PGG-Glucan treatment. Mobilization was not associated with the induction of alpha-chemokines, which have recently been reported to induce rapid progenitor cell mobilization. Competitive repopulation experiments performed in irradiated female C3H/HeN mice revealed that, at three months after transplantation, more male DNA was present in bone marrow, splenic, and thymic tissues from animals transplanted with cells obtained from mice 30 min after a 2 mg/kg PGG-Glucan dose than in tissues from animals transplanted with cells obtained from saline-treated mice. Additional experiments evaluated the mobilization effects of PGG-Glucan (2 mg/kg) administered to mice which had been pretreated for three consecutive days with G-CSF (125 microg/kg/day). When blood was collected 30 min after PGG-Glucan treatment, the number of GM-CFC mobilized in combination-treated mice was additive between the number mobilized in mice treated with G-CSF alone and the number mobilized in mice treated with PGG-Glucan alone. These studies demonstrate that: A) PGG-Glucan can rapidly mobilize PBPC; B) the kinetic pattern of PGG-Glucan-induced mobilization is different from that of the CSFs; C) the reconstitutional potential of PGG-Glucan mobilized cells is greater than that of steady-state PBPC, and D) PGG-Glucan can enhance G-CSF-mediated PBPC mobilization.

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2'-O-Methyl (2'OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2'OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2'OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application.

This report describes the safety observations following administration of a polyvalent DNA prime-protein boost HIV-1 vaccine formulated with adjuvant QS21. Local injection site reactions were the most common (65% of subjects), and included type IV delayed-type hypersensitivity (DTH) reactions at prior DNA inoculation sites in 12 of 28 (43%) subjects following protein vaccination. Systemic reactions revealed two cases of vasculitis temporally related to inoculation with recombinant Env protein+QS21 adjuvant. Questions remain regarding the cause of the vasculitis, but the unique DTH observation may have contributed to the high level of immune responses previously reported for this vaccine.