Increased vaccine tolerability and protection via NF-κB modulation.

Improving adjuvant responses is a promising pathway to develop vaccines against some pathogens (e.g., HIV or dengue). One challenge in adjuvant development is modulating the inflammatory response, which can cause excess side effects, while maintaining immune activation and protection. No approved adjuvants yet have the capability to independently modulate inflammation and protection. Here, we demonstrate a method to limit inflammation while retaining and often increasing the protective responses. To accomplish this goal, we combined a partial selective nuclear factor kappa B (NF-kB) inhibitor with several current adjuvants. The resulting vaccines reduce systemic inflammation and boost protective responses. In an influenza challenge model, we demonstrate that this approach enhances protection. This method was tested across a broad range of adjuvants and antigens. We anticipate these studies will lead to an alternative approach to vaccine formulation design that may prove broadly applicable to a wide range of adjuvants and vaccines.

Role of the L-amino acid transporter-1 (LAT-1) in mouse trophoblast cell invasion.

LAT-1 (L-type amino acid transporter 1) is a system L, Na(+)-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and it's over expression is related to tumor invasion. LAT-1 is highly expressed in the trophoblast giant cells (TGCs) at the time of implantation. Since trophoblast giant cells are highly invasive during the process of endometrial implantation and placentation, LAT-1 may play a role in the invasive phenotype. Our objectives were to identify the effects of increased and decreased LAT-1 expression on mouse trophoblast invasion. We therefore examined the role of amino acid deprivation, pharmacologic blockade specific to leucine transport and gene silencing (siRNA) on LAT-1 expression and trophoblast cell invasion. We utilized mouse primary trophoblast stem (TS) cells. LAT-1 mRNA expression was quantified by real time qPCR, protein by Western blotting and cell invasion was measured in Transwell plates through Matrigel. Amino acid transport using uptake of tritiated leucine. Under limited leucine availability and/or pharmacologic blockage, LAT-1 gene expression was significantly increased, p<0.05. This was associated with a 3-fold increase in cell invasion, p<0.05. In contrast, following siRNA-mediated gene silencing decreased LAT-1 expression (both mRNA and protein) was associated with decreased cell invasion and decreased leucine uptake, p<0.05. Upregulation of LAT-1 gene expression via limited amino acid availability or following pharmacologic blockade of transport leads to an increase in mouse trophoblast stem cell invasiveness. Downregulation of LAT-1 expression via genetic silencing leads to inhibition of invasiveness. These results demonstrate that LAT-1 plays an important role in trophoblast invasion.

LAT-1 expression in pre- and post-implantation embryos and placenta.

OBJECTIVES: LAT-1 (L-type amino acid transporter 1) is a system L, Na(+)-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and is related to tumor invasion. Primary rat hepatocytes in culture increase LAT-1 mRNA in response to amino acid depletion. Transformed hepatic cell lines demonstrate constitutive expression of LAT-1. These observations suggest that LAT-1 expression confers a growth and survival advantage under limited amino acid availability. LAT-1 is highly expressed in the placenta. It has been shown previously that amino acids are fundamental regulators of cell function and energy metabolism in pre-implantation embryos. Our objectives were to analyze qualitatively and quantitatively LAT-1 expression in pre-implantation stages of mouse embryo development and to identify cell types expressing LAT-1 in post-implantation stages. METHODS: LAT-1 was quantified by real-time qPCR. Localization of expression was by laser capture microdissection, in situ hybridization and immunohistochemistry. RESULTS: Our results show increasing mRNA levels of LAT-1 as the embryo develops from zygote to blastocyst with highest levels at hatching blastocyst. Expression studies of LAT-1 on microdissected samples from developing mouse placenta show highest levels of LAT-1 mRNA in trophoblast giant cells (TGCs) at the time of implantation (E7.5), followed by maternal decidua, ectoplacental cone and epiblast. At later stages of development (E9.5 and E11.5) no differential expression of LAT-1 was observed. In situ hybridization and immunohistochemistry also showed differential expression of LAT-1 mRNA and protein, respectively, with darkest staining in TGCs at E7.5. By E9.5 and E11.5 mRNA expression was no longer preferentially localized to TGCs, hybridization was equal across the different cell types and regions. LAT-1 protein expression, however, still showed highest intensity of staining in TGCs at E9.5 and E11.5. CONCLUSIONS: Since trophoblast giant cells are invasive cells that displace and phagocytose the uterine epithelial cells, these data suggest that LAT-1 may play a role in the invasive phenotype. The mechanism of LAT-1 regulation during placentation, therefore, might provide valuable clues to its role in tumor progression and invasion.

Liganded and unliganded steroid receptor modulation of beta 1 adrenergic receptor gene transcription.

The classical model of gene regulation by hormones involves a hormone-bound receptor interacting with a DNA response element to increase or decrease gene transcription. Steroid hormone regulation more commonly involves atypical cis-elements, co-receptors, accessory proteins, and unique modes of interaction on different genes. The thyroid hormone and retinoic acid receptors belong to the super family of steroid nuclear receptors and may modify gene expression even in the absence of ligand binding. In these studies, we characterized thyroid receptor- and retinoic acid receptor-mediated regulation of beta1 adrenergic receptor (beta1AR) gene expression. Using cloned fragments of the ovine beta1AR in a luciferase reporter vector, we examined the effects of thyroid receptor and retinoic acid receptor, alone and in combination with T3 or retinoic acid on beta1AR expression. We examined expression in SK-N-SH neuroblastoma cells, CV-1 fibroblasts, and, in neonatal rat, primary cardiomyocytes. We demonstrated that even in the absence of ligand binding, thyroid receptor and retinoic acid receptor can significantly increase beta1AR transcription activity. This effect is important in the developmental transition in beta1AR expression during fetal and postnatal life.

Beta-adrenergic receptors (betaAR) regulate cardiomyocyte proliferation during early postnatal life.

Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. beta-Adrenergic receptor (betaAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the betaAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of betaAR in rat cardiomyocyte growth in vivo. We demonstrated that betaAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of betaAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that betaAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin-treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after betaAR blockade. We conclude that the betaAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.

A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the beta1-adrenergic receptor gene.

The effects of glucocorticoids on expression of the beta1-adrenergic receptor (beta1AR) gene have been varied. To study the mechanism underling hormonal regulation of the beta1AR, transient transfection of progressively deleted ovine beta1AR promoter fragments was used to identify a 43-bp region (-1274 to -1232 from the translation start site) that contains a novel glucocorticoid regulatory unit (GRU) and confers glucocorticoid responsiveness. Using DNase I footprinting and electrophoretic mobility shift assays (EMSA), we demonstrated the GRU was composed of a palindrome, 5'-TAATTA-3', which is a core binding motif for the homeodomain proteins, an E-box (5'-CACGTG-3'), binding site for the Myc/Max family proteins, and an overlapping glucocorticoid response element (GRE) half-site (5'-TGTTCT-3'). EMSA demonstrated that the GRE half-site is critical for GRU-protein interactions, which also require binding of proteins to the E-box and the homeodomain region. Co-transfection of a plasmid expressing a c-myc antisense construct significantly reduced glucocorticoid responsiveness of the ovine beta1AR promoter. Furthermore, expression of proteins binding to the GRU was shown to be developmentally regulated, being high in embryonic, reduced in newborn and not detectable in adult heart. We conclude that the ovine beta1AR promoter contains a novel, functional GRU and that glucocorticoid receptor (GR) and the Myc/Max family proteins are involved in the cell-specific nuclear factor binding and transactivation via this element. The results suggest an alternative pathway through which glucocorticoids may exert their effects on genes lacking a full consensus GRE.

Drosophila awdK-pn, a homologue of the metastasis suppressor gene nm23, suppresses the Tum-1 haematopoietic oncogene.

The human nm23-H1 gene is a suppressor of solid tumour metastasis in some types of cancer. It is known that nm23 genes encode nucleoside diphosphate kinase polypeptides, but the regulatory pathways involving Nm23 are unclear. One approach to understanding nm23 function is to identify loci which interact with nm23. The Drosophila awd gene, a homologue of nm23, provides a model system for this genetic analysis. We report that the dominant awdK-pn allele suppresses haematopoietic defects associated with the Tum-l oncogene. Premature differentiation and aggregation of Tum-l blood cells is reduced by awdK-pn, resulting in an increased survival of Tum-l hemizygotes. Tum-l lethality is also suppressed by pn mutations, indicating the existence of a haematopoietic regulatory pathway involving the Tum-l, AwdK-pn and Pn proteins.