Oligodeoxynucleotides ODN 2006 and M362 Exert Potent Adjuvant Effect through TLR-9/-6 Synergy to Exaggerate Mammaglobin-A Peptide Specific Cytotoxic CD8+T Lymphocyte Responses against Breast Cancer Cells.

Mammaglobin-A (MamA) is overexpressed in 40-80% of all human breast cancers. Recent phase I clinical trials of the MamA DNA vaccine showed encouraging safety outcomes. However, this vaccine elicited only a modest increase in MamA specific CD8+T lymphocyte (CTL) activation. As vaccine adjuvants play a critical role in enhancing the immunotherapeutic efficiency of vaccines, we tested the potential role of three synthetic CpG oligodeoxynucleotides (ODN2216-class A ODN, ODN2006-class B ODN, and ODN M362-class C ODN) to further enhance MamA specific CTL responses. Towards this, naïve CD8+T cells were obtained from healthy HLA-A2+ human donors. The HLA-A2 specific immunodominant epitope of MamA, MamA2.1 (LIYDSSLCDL), was utilized to activate naïve CD8+T cells. The THP-1 (HLA-A2+) cells were used as antigen presenting cells to stimulate naïve CD8+T cells along with (or without) co-treatment of various ODNs mentioned above. Activation of naïve CD8+T cells with the MamA2.1 peptide along with ODNs demonstrated enhanced MamA specific CTL mediated cytotoxicity on AU565 (HLA-A+/MamA+) breast cancer cells following co-treatment with ODN2006 and M362 compared to ODN2216 or MamA2.1 peptide alone. However, no significant cytotoxicity was noted upon treatment of MamA2.1 activated CTLs on MCF7 (HLA-A+/MamA-) cells, suggesting that the activation of CTLs is specific to the MamA antigen. Functional characterization studies demonstrated specific IL-12 mediated cross-talk between TLR-6 and -9 in THP-1 cells following stimulation with ODN2006 and M362, which was critical for the final cytotoxic activation of CD8+T lymphocytes. Based on these data, we conclude that ODN2006 and ODN M362 exerted a strong adjuvant effect through induction of the initial innate immune response through TLR9 upregulation followed by enhanced MamA specific CTL dependent adaptive immune responses. Our current data provide evidence for the application of Class-B/-C-CpG-ODNs as potential vaccine adjuvants towards enhancing the success of MamA based breast cancer vaccination.

Dopaminergic DA1 signaling couples growth-associated protein-43 and long-term potentiation in guinea pig hippocampus.

The basic goal of the project was to determine whether dopaminergic DA1 receptor (DA1R) signaling couples growth-associated protein 43 (GAP-43; a putative "plasticity" protein) and long-term potentiation (LTP; an enduring form of synaptic plasticity). Thus, guinea pigs were prepped to stimulate the CA3 and evoke population spikes in the CA1 neurons in the hippocampus in vivo. Animals were injected with either saline or SCH23390 (a selective DA1R antagonist), 1-2 h prior to recordings. It was found that tetanic stimulation (100 Hz, 1 s, three trains at 15 s intervals) readily produced early-LTP and late-LTP in the saline group. In contrast, none of the guinea pigs pre-treated with SCH23390 developed late-LTP, though early-LTP had been present. Furthermore, both GAP-43 mRNA and protein were up-regulated after LTP induction in the saline group. However, GAP-43 protein up-regulation was blocked in animals treated with SCH23390. Anti-GAP-43 immunoreactivity was intense in CA3/CA1 synaptic regions, whereas GAP-43 mRNA hybridization was localized to somatic layers in the hippocampus. Altogether, our results suggest that dopaminergic DA1 signaling partly couples GAP-43 and LTP.

Cloning and partial characterization of four plasmalemmal-associated syntaxin isoforms in Limulus.

We describe herein the cloning of a group of syntaxins in Limulus that are associated with the plasma membrane. Initially, multiple degenerate oligonucleotide primers (DOP) and probes were designed from sequences of known plasma membrane associated syntaxins. Combined experiments using reverse transcriptase-polymerase chain reaction (RT-PCR), colony hybridization and reverse dot blot yielded three distinct probes. Subsequently, two cDNA libraries derived from the Limulus central nervous system (CNS) were screened and four distinct isoforms, designated Limulus syntaxin (Lim-syn) 1A, 1B, 1C and 1D, were obtained from forty cloned full-length sequences. The predicted amino acid (aa) sequences 1-265 were identical for Lim-syn 1A, 1C and for Lim-syn 1B, 1D, respectively. A comparison of the 265 aa cytoplasmic segments for the two subgroups Lim-syn 1A/1C and Lim-syn 1B/1D differed at 13 aa residues within this sequence. Lim-syn 1A and 1B contained 290 aa residues, and both contained a transmembrane domain (TMD, 267-288) and a myristylation-like site (286-290) at the C-termini. Lim-syn 1C (291 residues) contained only the TMD whereas Lim-syn 1D was truncated (277 residues) and had neither a TMD nor a myristylation-like site. All Lim-syn isoforms showed great identity with syntaxin 1-homologs (syntaxin 1A/1B) from various other species. Ribonuclease protection assay (RPA) analyses revealed distinctive expression patterns for individual Lim-syn transcripts but all were detectable in the CNS. Moreover, the antibody (anti-Lim-syn-1) produced against aa 133-145 epitope of Lim-syn identified a protein of approximately 35 kDa found only in CNS tissues.