Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

CpG DNA, a ligand for Toll-like receptor 9 (TLR9), has been one of the most promising immunotherapeutic agents. Although there are several types of potent humanized CpG oligodeoxynucleotide (ODN), developing "all-in-one" CpG ODNs activating both B cells and plasmacytoid dendritic cells forming a stable nanoparticle without aggregation has not been successful. In this study, we generated a novel nanoparticulate K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. In sharp contrast to K3 alone, K3-SPG stimulates human peripheral blood mononuclear cells to produce a large amount of both type I and type II IFN, targeting the same endosome where IFN-inducing D CpG ODN resides without losing its K-type activity. K3-SPG thus became a potent adjuvant for induction of both humoral and cellular immune responses, particularly CTL induction, to coadministered protein antigens without conjugation. Such potent adjuvant activity of K3-SPG is attributed to its nature of being a nanoparticle rather than targeting Dectin-1 by SPG, accumulating and activating antigen-bearing macrophages and dendritic cells in the draining lymph node. K3-SPG acting as an influenza vaccine adjuvant was demonstrated in vivo in both murine and nonhuman primate models. Taken together, K3-SPG may be useful for immunotherapeutic applications that require type I and type II IFN as well as CTL induction.innate immunity | two-photon microscopy | MARCO | Siglec-1 | β-glucan

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). Previous studies suggest that loss of AVP neurons might be the cause of polyuria in FNDI. Here we analyzed knockin mice expressing mutant NPII that causes FNDI in humans. The heterozygous mice manifested progressive polyuria as do patients with FNDI. Immunohistochemical analyses revealed that inclusion bodies that were not immunostained with antibodies for mutant NPII, normal NPII, or AVP were present in the AVP cells in the supraoptic nucleus (SON), and that the size of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) as well as in the nucleus of AVP neurons in 1-mo-old heterozygous mice. At 12 mo, dilated ER filled with aggregates occupied the cytoplasm of AVP cells, while few aggregates were found in the nucleus. Analyses with in situ hybridization revealed that expression of AVP mRNA was significantly decreased in the SON in the heterozygous mice compared with that in wild-type mice. Counting cells expressing AVP mRNA in the SON indicated that polyuria had progressed substantially in the absence of neuronal loss. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregates accumulated in the ER might be involved in the dysfunction of AVP neurons that lead to the progressive polyuria.

Neuropeptide Y (NPY) in the arcuate nucleus is an orexigenic hormone of which levels are regulated by humoral as well as neural signals. In this study, we examined the regulation of NPY gene expression in the arcuate nucleus in hypothalamic organotypic cultures. Dexamethasone (DEX) (10 Ϫ9 to 10 Ϫ7 M) significantly increased NPY mRNA expression, and the effects were not influenced by coincubation with the sodium channel blocker tetrodotoxin (TTX), indicating that the action of DEX is independent of action potentials. Conversely, insulin (10 Ϫ11 to 10 Ϫ9 M) significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was abolished in the presence of TTX. Because GABA and its receptors are expressed in the arcuate nucleus in vivo, we examined whether GABAergic systems were involved in the insulin action. The GABA B agonist baclofen significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was completely abolished in the presence of either the GABA A antagonist bicuculline or the GABA B antagonist CGP35348 ( p-3-aminopropyl-p-diethoxymethyl phosphoric acid). Furthermore, increases in the GABA-synthesizing enzyme glutamic acid decarboxylase 65 (GAD65) mRNA expression preceded decreases in NPY mRNA expression in the arcuate nucleus in the cultures. Experiments in vivo also demonstrated that increases in GAD65 mRNA expression in the arcuate nucleus preceded decreases in the NPY mRNA expression in a fasting-refeeding paradigm and that intracerebroventricular injection of insulin increased GAD65 mRNA expression in the arcuate nucleus in fasted rats. These data suggest that insulin inhibits NPY gene expression in the arcuate nucleus through GABAergic systems.

Ghrelin, which was identified from the rat stomach, is a potent stimulant for food intake. Several lines of evidence suggest that the orexigenic action of ghrelin is mediated via the neuropeptide Y (NPY) neurons in the arcuate nucleus, although the detailed mechanisms by which ghrelin stimulates NPY neurons are not clear. In this study, we examined the gene regulation of NPY and agouti-related peptide (AGRP), another orexigenic peptide synthesized in the NPY neurons, in the arcuate nucleus by ghrelin in hypothalamic organotypic cultures. Incubation of the hypothalamic explants with ghrelin significantly increased NPY and AGRP mRNA expression in the presence, but not absence, of dexamethasone. Glucocorticoids were also necessary for ghrelin action in vivo because an intracerebroventricular injection of ghrelin significantly increased NPY and AGRP mRNA expression in the arcuate nucleus only in sham-operated, but not in adrenalectomized rats. The stimulatory effects of ghrelin on gene expression were not blocked by a sodium channel blocker tetrodotoxin in the organotypic cultures. Ghrelin also increased NPY heteronuclear (hn) RNA expression, the first transcript that has been used as an indicator for gene transcription. The stimulatory effects of ghrelin on NPY gene expression were abolished in the presence of cycloheximide, which blocks translation, suggesting that de novo protein synthesis is required for ghrelin action. These data suggest that ghrelin stimulates NPY and AGRP gene expression independently of action potentials only in the presence of glucocorticoids. Furthermore, our data demonstrate stimulatory action of ghrelin on NPY gene transcription, which requires de novo protein synthesis.

Mechanical stresses regulate physiological and pathological functions of vascular endothelial cells. We examined, in this study, the effects of hypergravity on endothelial functions. Hypergravity (3 G) applied by low speed centrifuge immediately induced a membrane translocation of small G-protein RhoA and tyrosine phosphorylation of 125 kDa FAK in bovine aortic endothelial cells (BAECs). Hypergravity also induced a transient reorganization of actin fibers in 3 min, which was inhibited by Rho-kinase inhibitor (Y27632) and tyrosine kinase inhibitors (herbimycin A and tyrphostin 46). Furthermore, the extracellular ATP concentration ([ATP]o) was increased by 2 G and 3 G hypergravity in 5 min, and the inhibitors of Rho-kinase, tyrosine kinase, and volume-regulated anion channels (VRAC; verapamil, tamoxifen and fluoxetine) significantly suppressed [ATP]o elevation. Application of 3 G hypergravity for 1 h increased the nuclear uptake of BrdU, which was inhibited by Rho-kinase inhibitor and VARC inhibitors. Furthermore, intermittent application of 3 G hypergravity for 1 or 2 h/day stimulated endothelial migration in 5 days, and this was inhibited by suramin, a P2 antagonist. Collectively, these results indicate that hypergravity induces ATP release and actin reorganization via RhoA activation and FAK phosphorylation, thereby activating cell proliferation and migration in BAECs. These also suggest that gravity can be regarded as an extracorporeal signal that could significantly affect endothelial functions.

Advax, a delta inulin-derived microparticle, has been developed as an adjuvant for several vaccines. However, its immunological characteristics and potential mechanism of action are yet to be elucidated. Here, we show that Advax behaves as a type-2 adjuvant when combined with influenza split vaccine, a T helper (Th)2-type antigen, but behaves as a type-1 adjuvant when combined with influenza inactivated whole virion (WV), a Th1-type antigen. In addition, an adjuvant effect was not observed when Advax-adjuvanted WV vaccine was used to immunize toll-like receptor (TLR) 7 knockout mice which are unable to respond to RNA contained in WV antigen. Similarly, no adjuvant effect was seen when Advax was combined with endotoxin-free ovalbumin, a neutral Th0-type antigen. An adjuvant effect was also not seen in tumor necrosis factor (TNF)-α knockout mice, and the adjuvant effect required the presences of dendritic cells (DCs) and phagocytic macrophages. Therefore, unlike other adjuvants, Advax potentiates the intrinsic or in-built adjuvant property of co-administered antigens. Hence, Advax is a unique class of adjuvant which can potentiate the intrinsic adjuvant feature of the vaccine antigens through a yet to be determined mechanism.

We report that deamination coupled with 5-carboxyvinyldeoxyuridine-mediated photobranching causes the heat-induced transition of cytosine to uracil with high efficiency without any side reaction.