Effect of cigarette smoke on mucosal vaccine response with activation of plasmacytoid dendritic cells: The outcomes of in vivo and in vitro experiments.

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.

Treatment with Lactobacillus Retards the Tumor Growth of Head and Neck Squamous Cell Carcinoma Cells Inoculated in Mice.

Bacteria have been used for more than a century to treat solid tumors. Because solid tumors generate an anaerobic environment, we evaluated the anti-tumor effect of the obligate anaerobe strain KK378, derived from Lactobacillus casei (L. casei), using mice bearing head and neck cancer. Wild-type L. casei is a nonpathogenic bacterium that is commonly used in foods. Moreover, patients with head and neck squamous cell carcinoma often have multiple cancers and cervical lymph node metastasis that can be directly sensed beneath the skin. To establish the animal model bearing head and neck cancer, we inoculated each of human squamous cell carcinoma cell lines, SAS, HSQ89, and HSC2, on the back skin of BALB/cSlc-nu/nu mice. After tumor formation, L. casei KK378 was administered directly into the tumor, and tumor size and serum cytokine levels were analyzed. Mice injected with 108 cfu of L. casei KK378 showed reduction in tumor growth compared with PBS control; especially, the SAS tumor was significantly reduced (p = 0.008). Administered L. casei KK378 was detected in tumor tissues but not in normal tissues (liver, kidney, and lung) of SAS tumor-bearing mice, which was associated with increased blood cytokines (TNF-α, IFN-γ, IL-5, IL-10, and IL-12). Among these cytokines, the serum levels of IFN-γ and TNF-α were significantly increased (p < 0.05). In conclusion, L. casei KK378 infection may suppress tumor growth by inducing the host immune response. Direct injection of Lactobacillus into the tumor could be a potential strategy to treat head and neck squamous cell carcinoma.

Hyperoxia causes diffuse alveolar damage through mechanisms involving upregulation of c-Myc/Bax and enhanced production of reactive oxygen species.

BACKGROUND: Hyperoxia is a known cause of diffuse alveolar damage (DAD). We previously reported the transcript profiling of DAD induced by hyperoxia exposure in mouse lungs and showed that the gene expression of myelocytomatosis oncogene (c-Myc) was significantly upregulated whereas that of surfactant-associated protein (SP)-C was downregulated. However, the mechanism underlying hyperoxia-induced DAD is not well understood. METHODS: The hyperoxia-induced changes in SP-A/B/C/D, c-Myc, B-cell chronic lymphocytic leukemia/lymphoma (Bcl)-2, and Bcl-2-associated X protein (Bax) expression in mouse lungs were examined by cDNA microarray analysis. The expression levels of the above mentioned genes, cell viability, caspase activity, and reactive oxygen species (ROS) production were also examined in the human lung adenocarcinoma cell line A549 and mouse fibroblast-like cell line NIH/3T3. RESULTS: Hyperoxia induced a decrease in SP-C/A expression in mouse lungs, and SP-C downregulation was also confirmed in A549 cells. In addition to enhanced c-Myc expression, Bax expression also increased following exposure of the mice to hyperoxia. In vitro analysis showed that expression of these genes is regulated in a cell-type-dependent manner, i.e., upregulation of c-Myc in NIH/3T3 cells and Bax in A549 cells occurred regardless of whether there was a similar decrease in cell viability and increase in caspase-3/7 activation in response to hyperoxia. ROS production and caspase-8 activation were also observed in both cells. CONCLUSIONS: We concluded that hyperoxia induces ROS production and cell death in lung tissues through a cell-type specific mechanism involving the upregulation of c-Myc/Bax, and caspase-8 and -3/7 activation-dependent pathways, thereby leading to the development of DAD.

A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H)1 immunity.

BACKGROUND: CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated T(H)1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H)1 and T(H)2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H)1, but not T(H)2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H)1 immunity.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Identification of cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) as a novel alpha1A-adrenoceptor-down-regulating protein and establishment of an alpha1L-adrenoceptor-expressing cell line.

Two distinct alpha(1)-adrenoceptor phenotypes (alpha(1A)- and alpha(1L)-ARs) are known to originate from a single ADRA1A(alpha(1a)) gene by an as-yet-unknown mechanism. We hypothesized that an alpha(1a)-AR-interacting protein could generate the alpha(1L)-AR phenotype and we sought to identify such a protein and to examine its effects on the expression of alpha(1A) and alpha(1L) phenotypes. Cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) was first identified using a yeast two-hybrid approach as an alpha(1a)-AR-interacting protein. Transfection of alpha(1a)-AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing alpha(1A)-ARs having a predominant high affinity site for prazosin, with a low proportion (<10%) of prazosin-low affinity sites (alpha(1L)-AR). Knockdown of endogenous CHO-CRELD1alpha [alpha(1a)-CKD(alpha(1A)-enhanced) cells] enhanced the expression of alpha(1A)-AR, whereas over-expression of CRELD1alpha reduced alpha(1A)-AR expression, yielding alpha(1a)-COE(alpha(1L)-dominant) cells expressing a high proportion (50%) of the alpha(1L)-AR phenotype. The ligand binding and functional agonist and antagonist profiles in alpha(1a)-CKD(alpha(1A)-enhanced) and alpha(1a)-COE(alpha(1L)-dominant) cell lines were entirely in accord with the alpha(1A)-AR and alpha(1L)-AR phenotypes observed in intact tissues. CRELD1alpha down-regulates expression of the alpha(1A)-AR, thereby enhancing the proportion of expression of the alpha(1L)-AR phenotype. The alpha(1L)-AR-expressing alpha(1a)-COE(alpha(1L)-dominant) cell line reflects accurately the phenotype of this AR observed in vivo and will facilitate development of alpha(1L)-AR-targeted drugs.

Snapin, a new regulator of receptor signaling, augments alpha1A-adrenoceptor-operated calcium influx through TRPC6.

Activation of G(q)-protein-coupled receptors, including the alpha(1A)-adrenoceptor (alpha(1A)-AR), causes a sustained Ca(2+) influx via receptor-operated Ca(2+) (ROC) channels, following the transient release of intracellular Ca(2+). Transient receptor potential canonical (TRPC) channel is one of the candidate proteins constituting the ROC channels, but the precise mechanism linking receptor activation to increased influx of Ca(2+) via TRPCs is not yet fully understood. We identified Snapin as a protein interacting with the C terminus of the alpha(1A)-AR. In receptor-expressing PC12 cells, co-transfection of Snapin augmented alpha(1A)-AR-stimulated sustained increases in intracellular Ca(2+) ([Ca(2+)](i)) via ROC channels. By altering the Snapin binding C-terminal domain of the alpha(1A)-AR or by reducing cellular Snapin with short interfering RNA, the sustained increase in [Ca(2+)](i) in Snapin-alpha(1A)-AR co-expressing PC12 cells was attenuated. Snapin co-immunoprecipitated with TRPC6 and alpha(1A)-AR, and these interactions were augmented upon alpha(1A)-AR activation, increasing the recruitment of TRPC6 to the cell surface. Our data suggest a new receptor-operated signaling mechanism where Snapin links the alpha(1A)-AR to TRPC6, augmenting Ca(2+) influx via ROC channels.

[Alpha1-adrenoceptor subtypes and alpha1-adrenoceptor antagonists].

Alpha(1)-adrenoceptors are widely distributed in the human body and play important physiologic roles. Three alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) have been cloned and show different pharmacologic profiles. In addition, a putative alpha(1)-adrenoceptor (alpha(1L) subtype) has also been proposed. Recently, three drugs (tamsulosin, naftopidil, and silodosin) have been developed in Japan for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. In this review, we describe recent alpha(1)-adrenoceptor subclassifications and the pharmacologic characteristics (subtype selectivity and clinical relevance) of alpha(1)-adrenoceptor antagonists.