The distribution and fine structure of lymphatic vessels associated with nerves was studied by immunohistochemistry in the murine craniofacial region. The tissue sections and blocks were immunostained for LYVE-1, protein gene product 9.5, CD34 and aquaporin-1 to demonstrate the lymphatic vessels, nerves, blood vessels and water channel protein, respectively. Transmission electron microscopic examination was also performed to investigate the relationship between the lymphatics and nerves. In the nasal area, the lymphatics were found in dura mater on the cribriform plate and beneath the nasal mucosa, this supposedly supplying the cerebrospinal fluid drainage route along the olfactory nerves. The proximal portions of the cranial nerves were equipped with the lymphatics in the epineurium. In the distal portions of the nerves, the lymphatics were distributed in close proximity of the perineural sheath, and thus might contribute to maintenance of microenvironment suitable for the nerves by an absorptive activity of the lymphatic endothelial cells. The present findings suggest that the lymphatic system associated with the cranial nerves provides the pathway for transport of cerebrospinal fluid, tissue fluid, and free cells involved in immune response and tumor metastasis in the craniofacial region.

Advanced solid tumors are exposed to hypoxic conditions over longer periods of time as they grow. Tumor hypoxia is a major factor that induces malignant progression, but most previous studies on tumor hypoxia were performed under short‐term hypoxia for up to 72 hours and few studies have focused on tumor response to chronic hypoxic conditions. Here we show a molecular mechanism by which chronic hypoxia promotes invasive behavior in prostate cancer cells. We found that an epithelial‐mesenchymal transition (EMT)‐driving transcription factor, slug, is specifically upregulated under chronic hypoxia and promotes tumor cell migration and invasion. Unexpectedly, processes associated with EMT, such as loss of E‐cadherin, are not observed under chronic hypoxia. Instead, expression of ephrin‐B1, a ligand of Eph‐related receptor tyrosine kinases, is markedly induced by slug through E‐box motifs and promotes cell migration and invasion. Furthermore, slug and ephrin‐B1 are highly coexpressed in chronic hypoxic cells of human prostate adenocarcinoma tissues after androgen deprivation, which is known to cause tumor hypoxia. Taken together, these results indicate that chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating the expression of ephrin‐B1. In addition, ephrin‐B1 may be a novel therapeutic target in combination with androgen deprivation therapy for aggressive prostate cancer.

The cellular distribution of 5'-nucleotidase (5'-Nase) was studied in the rat tissues at the levels of protein and mRNA by use of immunohistochemistry and in sift: hybridization, and compared with that of reaction products by enzyme-itistochemistiy. 5'-Nase activity, immunoreactivity of ecto-5'-Nase (CD73) and hybridization signals for its 1nRNA were colocalized in the lymphatic vessels including central lacteals of the small intestine, suggesting that 5'-Nase is actually produced in the lymphatic endothelial cells and allocated to their cell membrane as an enzyme to regulate lymph production and flow. Double immunostainig for CD73 and Factor VIII-related antigen clearly showed the distinction and relationship between lymphatics and the blood vessels. The present findings support our view that 5'-Nase is an available marker of lymphatics, and indicate the usefulness of the histochemical methods for 5'-Nase not only for demonstration of lymphatics, but also for examination of the functional roles a11d dynamics of 5'-Nase in the lymphatic endothelial cells in physiological and pathological conditions.

The intestinal mucosa is vulnerable to an ischemia-reperfusion (I/R) attendant on some bowel diseases and surgery; thus, the restoration of the mucosal integrity is critical to achieving functional recovery of the intestine injured by I/R. In this histochemical study, we investigated the alteration of the central lacteals--which are essential for the transport of fat, tissue fluid, and immune cells in the intestinal mucosa--in the murine jejunum after I/R. The intestine inflicted with I/R demonstrated mucosal injury involving the inflammatory response, with interstitial edema, disruption of the villous tissue, and subsequent tissue regeneration of the villi. The regenerative villous tissue revealed lymphatic regrowth showing proliferative activity from the residual mucosal lymphatics behind the regenerated blood vasculature. During the regenerative phase, the blood vascular pericytes expressed an intense immunoreaction for VEGF-A, an inducer for monocyte/macrophage recruitment as well as angiogenesis. Also, the F4/80-immunopositive macrophages significantly increased in number in the regenerating villous stroma. Furthermore, the macrophages recruited around the regrowing lacteals expressed the immunoreactivity for VEGF-C, which is a highly specific lymphangiogenic factor. The present study is first to delineate alterations in the central lacteals in the small intestine following I/R, thereby suggesting that the recruitment of the macrophages induced by upregulation of VEGF-A in the pericytes of regenerative blood vessels might promote reconstruction of the central lacteals through their release of VEGF-C.

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The cellular expression of neurokinin 1 (NK1) receptor in the rat duodenal villi was studied at the levels of protein and mRNA by use of immunohistochemistly and in sin: hybridization. Transmissio11 electron microcopy was also performed to investigate the functional significance of the cells expressing NK1 receptor. Both the NK1 receptor-immunoreactivity and hybridization signals for its mRNA were localized in the fibroblast-like cells beneath the villous epithelium, indicating that NK1 receptor is produced and allocated in the cells. These cells connected with each other by their slender processes to form a well-developed network, which is probably provided with a contractile potential by the cellular characteristics including an abundance of cytoplasmic myofilaments and gap junctions between the processes, throughout the lamina propria of the villi. They were intimately associated with blood vessels and nerve fibers containing substance P. The present findings suggest that the contractile network of the fibroblast-like cells may regulate the blood flow of the vascular network in the villi by interaction between substance P released from the nerve terminals and its receptor on the cells.Tachykinin peptides are well known to be neurotransmitters extensively distributed throughout the mammalian enteric nervous system (4, 24, 28). The preprotachykinin-A gene-derived peptides, substance P and neurokinin A, have been reported to exert variable effects on gastrointestinal activities, including motility, fluid secretion and vascular and immune functions (ll), 22).The actions of the neuropeptides are mediated by specific, high-affinity G-protein-coupled receptors (19). Three types of tachykinin receptors, termed neurokinin (NK)l, NK2 and NK3 receptors, have been described by pharmacological (6, 7, ll, 14, 15) and biochemical studies (9, 23). Several immunohistochemical studies indicate that NKI receptor, being preferred receptor for substance P, is localized on subpopulation of intramural neurons and interstitial cells of Cajal (lCCs) at the level of deep muscular Correspondence to: H. Shimoda at the above address. Tel and Fax: +81-97-586-5623 E-mail: hshimoda@oita-med.ac.jp nerve plexus in the intestines of mouse (32), rat (5, 27, 31) and guinea pig (12, 13, 20). Vannuchi and Faussone-Pellegrini (32) recently reported that NKI receptor-immunoreactivity was found on non-neuronal cells in the ileal villi only of mouse, but not of rat and guinea pig. Thus, it remains undetermined whether NKI receptor is localized i11 cellular elements of the intestinal mucosa in animals other than mouse.The present study here demonstrates the precise cellular expression of NKl receptor in the rat duodenal villi by immunohistochcmistry, in sift: hybridization and electron microscopy, and its functional significance is also discussed. MATERIALS AND METHODSSeven adult Wistar rats of both sexes (l50-25O g) were used in this study. They were given standard laboratory animal chow and water ad Iibimrn under routine laboratoiy conditi...

Helicobacter pylori infection is the strongest known risk factor of stomach cancer. Strains harboring the virulence factor CagA (cytotoxin-associated gene A) significantly stimulate host inflammatory response, which increases the risk of ulceration and cancer. However, the mechanisms by which CagA triggers prolonged inflammation with mucosal damage remain elusive. Based on a large-scale genetic screen using Drosophila, we identified a novel CagA target Synaptotagmin-like protein 2-a, Slp2-a, an effector of small GTPase Rab27. Using gastric organoid-derived monolayers of polarized mucous cells, we demonstrated that CagA inhibited Slp2-a-mediated docking of mucous granules to the plasma membrane by direct binding to Slp2-a. We further observed aberrant cytoplasmic retention of mucus in human gastric mucosa infected with CagA-expressing strains. These results suggest that CagA could be disrupting the protective mucous barrier by inhibiting Slp2-a-mediated mucous granule exocytosis, which may lead to mucosal damage from luminal acid and pepsin to promote inflammation leading to cancer.