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Importin α1/KPNA1 is a member of the Importin α family widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. In humans, a de novo mutation of the KPNA1 (human Importin α5) gene has been linked with schizophrenia; however, the precise roles of KPNA1 in disorder-related behaviors are still unknown. Moreover, as recent studies have highlighted the importance of gene-environment interactions in the development of psychiatric disorders, we investigated the effects of Kpna1 deletion and social isolation stress, a paradigm that models social stress factors found in human patients, on psychiatric disorder-related behaviors in mice. Through assessment in a behavioral battery, we found that Kpna1 knockout resulted in the following behavioral phenotype: (1) decreased anxiety-like behavior in an elevated plus maze test, (2) short term memory deficits in novel object recognition test (3) impaired sensorimotor gating in a prepulse inhibition test. Importantly, exposure to social isolation stress resulted in additional behavioral abnormalities where isolated Kpna1 knockout mice exhibited: (1) impaired aversive learning and/or memory in the inhibitory avoidance test, as well as (2) increased depression-like behavior in the forced swim test. Furthermore, we investigated whether mice showed alterations in plasma levels of stress-associated signal molecules (corticosterone, cytokines, hormones, receptors), and found that Kpna1 knockout significantly altered levels of corticosterone and LIX (CXCL5). Moreover, significant decreases in the level of prolactin were found in all groups except for group-housed wild type mice. Our findings demonstrate that Kpna1 deletion can trigger widespread behavioral abnormalities associated with psychiatric disorders, some of which were further exacerbated by exposure to adolescent social isolation. The use of Kpna1 knockout mice as a model for psychiatric disorders may show promise for further investigation of gene-environment interactions involved in the pathogenesis of psychiatric disorders.

Psychological stress is deeply involved in the pathophysiology of not only mental illness but also functional gastrointestinal disorders. In the present study, we examined the relationship between psychological stress and abnormality of stool properties, focusing on the alteration of plasma glucocorticoid and guanylin (GN)/uroguanylin (UGN) expression in the colon. A murine model of chronic social defeat stress (CSDS) was established by exposing a C57BL/6N intruder mouse to a CD-1 aggressor mouse for 3–5 min. Thereafter the mice were kept in the same cage but separated by a divider for the remainder of the day. This procedure was repeated for 10 consecutive days, and then a social interaction test was performed to evaluate social avoidance. Fresh fecal and blood samples were collected for stool property analysis and measurement of the plasma glucocorticoid level by ELISA. The expression of GN, UGN, and guanylate cyclase 2C in the colonic tissues was examined by real-time RT-PCR and immunohistochemistry. Moreover, Lovo cells were stimulated with dexamethasone, and the expression of GN/UGN mRNA was examined. In the CSDS group, the time spent in the social interaction zone was significantly shorter when the CD-1 aggressor mouse was present than when it was absent. The social interaction ratio was also significantly lower in the CSDS group relative to the controls. The mean Bristol scale score was significantly lower in the CSDS group, but the fecal sodium concentration did not differ between CSDS mice and controls. The level of plasma corticosterone was significantly higher in the CSDS group than in the controls immediately after the 10th day of CSDS. The expression of both GN and UGN was significantly decreased in the CSDS mice. GN was expressed in all colonic epithelial cells, and UGN was expressed in ovoid or pyramidal epithelial cells in the colonic mucosa. The expression of both GN and UGN was significantly decreased in the CSDS mice relative to controls. The expression of both GN and UGN was significantly suppressed in Lovo cells upon stimulation with dexamethasone. Psychological stress-induced glucocorticoid may suppress colonic GN/UGN expression, resulting in a change in stool properties leading to constipation.

To elucidate the possible involvement of nitric oxide (NO) derived from inducible NO synthase (iNOS) in the pathogenesis of patients with allergic rhinitis, we used an animal model of atopic dermatitis (AD) induced by epicutaneous sensitization and analysed the differences in ear thickness, the frequency of scratching and plasma levels of ovalbumin-specific immunoglobulin E (OVA-IgE), transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) between control and iNOS(-/-) mice. Eight-week-old control and iNOS(-/-) male C57BL/6j mice were sensitized three times with OVA antigen. Before and after the last skin sensitization, the number of scratching incidents and the thickness of the ear were examined, and the plasma levels of OVA-IgE, α-MSH, ACTH, TGF-β and TNF-α were analysed by ELISA. Sensitization of mice with OVA resulted in increased plasma levels of OVA-IgE, α-MSH, ACTH, TGF-β and TNF-α in control, but not in iNOS(-/-) mice. The administration of l-nitro-arginine-methyl ester (l-NAME) abolished all the above changes that occurred in the control mice. In addition, iNOS(-/-) mice given α-MSH exhibited a change similar to that seen in the control, whereas iNOS(-/-) mice given ACTH, TGF-β or TNF-α did not demonstrate any changes. These results indicate that symptoms of AD such as scratching can be exacerbated by α-MSH, which is induced by iNOS-derived NO.

Because AA (L-ascorbic acid) scavenges various types of free radicals to form MDAA (monodehydroascorbic acid) and DAA (dehydroascorbic acid), its regeneration from the oxidized metabolites is critically important for humans and other animals that lack the ability to synthesize this antioxidant. To study the dynamic aspects of AA metabolism in the circulation, a long acting AOase (ascorbate oxidase) derivative was synthesized by covalently linking PEG [poly(ethylene glycol)] to the enzyme. Fairly low concentrations of the modified enzyme (PEG–AOase) rapidly decreased AA levels in isolated fresh plasma and blood samples with a concomitant increase in their levels of MDAA and DAA. In contrast, relatively high doses of PEG–AOase were required to decrease the circulating plasma AA levels of both normal rats and ODS (osteogenic disorder Shionogi) rats that lack the ability to synthesize AA. Administration of 50 units of PEG–AOase/kg of body weight rapidly decreased AA levels in plasma and the kidney without affecting the levels in other tissues, such as the liver, brain, lung, adrenal grand and skeletal muscles. PEG–AOase slightly, but significantly, decreased glutathione (GSH) levels in the liver without affecting those in other tissues. Suppression of hepatic synthesis of GSH by administration of BSO [L-buthionin-(S,R)-sulfoximine] enhanced the PEG–AOase-induced decrease in plasma AA levels. These and other results suggest that the circulating AA is reductively regenerated from MDAA extremely rapidly and that hepatic GSH plays important roles in the regeneration of this antioxidant.

Microglia are the main resident immunocompetent and phagocytic cells in the central nervous system (CNS). Activated microglia could play phagocytic roles as well as mediate inflammatory processes in the CNS. Involvement of activated microglia in the pathogenesis has been demonstrated in several neurological diseases including Alzheimer's disease (AD). Juzen-taiho-to (JTT), a traditional herbal medicine, has been reported to have effects on activating immune responses and phagocytosis. So far, little is known about the effects of this Kampo formulation JTT on microglia and in AD. In this report, we studied the effects of JTT on the activation and phagocytic functions of mouse microglia and bone marrow-derived macrophages (BMM). JTT could activate microglia, which was confirmed by the prominent morphological change and increased surface expression of an activation marker CD11b. In addition, JTT was revealed to induce microglial proliferation, and enhance microglial phagocytosis of, without eliciting an excessive production of nitric oxide. Furthermore, when mice were administrated with JTT in vivo, their BMM showed more effective phagocytosis of fibrillar Abeta(1-42). These findings implicate the therapeutic potential of JTT in AD and other neurological diseases accompanied by microglial activation.

The role of interleukin (IL)-18 in the protection from interstitial pneumonia and pulmonary fibrosis induced by bleomycin (BLM) was investigated by comparing the severity of BLM-induced lung injuries between wild-type and C57BL/6 mice with a targeted knockout mutation of the IL-18 gene (IL-18−/− mice). IL-18−/− mice showed much worse lung injuries than wild-type mice, as assessed by the survival rate, histological images, and leukocyte infiltration in the bronchoalveolar lavage fluid and myeloperoxidase activity. In wild-type mice, administration of IL-18 before BLM instillation resulted in suppression of lung injuries, increases in the hydroxyproline content, and decreases in the granulocyte-macrophage colony-stimulating factor content in the lung. Preadministration of IL-18 also resulted in prevention of the reduction of the lung IL-10 content caused by BLM-induced damage of alveolar epithelial. BLM instillation suppressed superoxide dismutase (SOD) activity in IL-18−/− mice to a greater extent than in wild-type mice. Pretreatment of IL-18 augmented Mn-containing superoxide dismutase (Mn-SOD) messenger RNA expression and SOD activity in the lung and prevented the reduction of SOD activity caused by BLM in both wild-type and IL-18−/− mice. These results suggest that IL-18 plays a protective role against BLM-induced lung injuries by upregulating a defensive molecule, Mn-SOD.

SummaryPsychological/physical stresses are known to cause relapses of autoimmune and inflammatory diseases. To reveal a mechanism by which noninflammatory stresses affect host defenses, responses to immobilization stress in mice were investigated, focusing on the role of a multifunctional cytokine, interleukin-18 (IL-18). In the adrenal cortex, the stress induced IL-18 precursor proteins (pro-IL-18) via adrenocorticotropic hormone (ACTH) and a superoxide-mediated caspase-1 activation pathway, resulting in conversion of pro-IL-18 to the mature form, which was released into plasma. Inhibitors of caspase-1, reactive oxygen species, and P38 mitogen-activated protein kinase (MAPK) suppressed stress-induced accumulation of plasma IL-18. These inhibitors also blocked stress-induced IL-6 expression. This, together with the observation that IL-6 was not induced in IL-18-deficient mice, showed that IL-6 induction by stress is dependent on IL-18. In stressed organisms, IL-18 may influence pathological and physiological processes. Controlling the caspase-1 activating pathway to suppress IL-18 levels may provide preventative means against stress-related disruption of host defenses.

Psychological/physical stresses have been reported to exacerbate auto-immune and inflammatory diseases. To clarify a mechanism by which non-inflammatory stresses disrupt host defenses, responses to immobilization stress in mice were investigated, focusing on the role of a multifunctional cytokine, interleukin-18 (IL-18). In the adrenal cortex, the stress induced IL-18 precursor proteins (pro-IL-18) via ACTH and a superoxide-mediated caspase-1 activation pathway, resulting in conversion of pro-IL-18 to the mature form which was released into plasma. Inhibitors of caspase-1, reactive oxygen species and P38 MAPK prevented stress-induced accumulation of plasma IL-18. These inhibitors also blocked stress-induced IL-6 expression. This, together with the observation that IL-6 was not induced in stressed-IL-18 deficient mice, showed that IL-6 induction by stress is dependent on IL-18. In stressed organisms, IL-18 may influence pathological and physiological processes. Controlling the caspase-1 activating pathway to suppress IL-18 levels may provide preventative means against stress-related disruption of host defenses.

Objective: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. Methods/Results: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R-/-) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R+/- mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R+/-, but not of MC2R-/-, mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R+/-, but not of MC2R-/-, mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. Conclusion: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

To estimate the potent immunomodulating effects of different types of traditional Japanese millet, we analyzed the effect of bran extracts of foxtail millet (Awa in Japanese), barnyard millet (Hie) and proso millet (Mochi-kibi) on nitric oxide (NO) and inflammatory cytokine production induced by lipopolysaccharide (LPS) in a mouse macrophage cell line (RAW264.7 cells). All methanol extracts of these millet brans showed suppressive activities against the production of NO and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 in LPS-stimulated macrophages, which were not responsible for their cytotoxic activities. These immunosuppressive activities were roughly proportional to the contents of the phenolic compounds in their extracts. Especially, the extract of proso millet exhibited the strongest immunosuppressing effect, which was associated with the highest content of phenolic compound. However, the extracts did not exhibit significant suppressive effects on the production of an anti-inflammatory cytokine, IL-10, in the same macrophage culture system. These results suggest that traditional Japanese millets have potent immunomodulating activities against the production of NO and cytokine production in activated macrophages.

Interleukin-18 (IL-18) is a pleiotropic cytokine expressed in both immune and non-immune cells. In the present study, we demonstrate an anti-apoptotic role of IL-18 in normal human neonatal foreskin epidermal keratinocytes (NHEK-F). Cultured NHEK-F spontaneously produced the active form of IL-18. Treatment of NHEK-F cells with anti-IL-18 receptor alpha-chain neutralizing antibody increased apoptosis and caspase-3 activity. Exogenous IL-18 augmented phosphorylation of Akt and activation of NF-kappaB. The promotion of Akt phosphorylation by IL-18 was abolished by LY294002, a PI3K inhibitor, but not SN50, an NF-kappaB inhibitor, indicating that IL-18 functions via the PI3K/Akt pathway and independently of NF-kappaB. In addition, IL-18 was found to augment expression of anti-apoptotic proteins, Bcl-2, XIAP and glucose regulated protein78/BiP, while anti-IL-18 receptor alpha-chain neutralizing antibody suppressed expression of Bcl-2, XIAP, glucose regulated protein94 and protein disulfide isomerase. Taken together, these results indicate that IL-18 plays an important role in keratinocyte survival.

Functional defect of cytoskeletal systems in the brain is to be elucidated to clarify the pathological process of neurodegenerative disorders such as Alzheimer’s disease (AD). Unlike microtubule or microfilament, tissue-specific intermediate filament exists in the central nervous system. Vimentin, glial fibrillary acidic protein (GFAP), and neurofilament proteins are localized in mesenchymal cell, astrocyte and neuron, respectively. Neurofilament is heteropolymer composed of triplet proteins; NF-L, NF-M, and NF-H, all phosphorylated proteins transported on a slow component of axonal flow. Phosphorylation of the rod domain of NF-L inhibits polymerization of neurofilament, and phosphorylation of NF-M, and NF-H regulates interaction with other cytoskeletal proteins, organellas, and membrane. Axonal neurofilaments are more phosphorylated than those in perikarya and dendrites. Phosphorylation of cytoskeletal protein should be closely related with the neurodegenerative process. In this chapter the aberration of cytoskeletal proteins in the brain of model animals for AD is discussed. Change in neurofilament was studied under different kinds of animal models which were designed to represent each specific aspect of pathogenetic process of AD. Abnormal accumulation of phosphorylated neurofilament protein is observed under various conditions of neuronal degeneration. Traumatic brain injury induces accumulation of neurofilament protein in neuronal perikarya at the impact site.

Change in calcium response was studied to clarify the pathological process of Alzheimer''s disease (AD). Cultured fibroblasts from patients with familial Alzheimer''s disease (FAD; n = 6), sporadic Alzheimer’s disease (SAD; n = 4), and age-matched healthy control subjects (n = 4) were studied with an ACAS Interactive Laser Cytometer (ACAS-470). Fibroblasts from two independent families with FAD (OS-1, and OS-2 families) showed a suppressed calcium response after stimulation by 100 nM bradykinin (BK) 100 nM Vasopressin (VP)or 10% FCS in Ca2+-free condition compared with control fibroblasts at 48 h after plating. However, on the 7th day after plating, the abnormal calcium response was no longer observed. The height of the calcium peak showed periodic variation, indicating a relationship of calcium response with the cell cycle. When fibroblasts from OS-1 and OS-2 families were arrested in S phase, they showed a significantly suppressed calcium peak after BK stimulation. However, when those fibroblasts were arrested in other phases, they showed the same calcium peak as the other cells. The suppression of calcium response in S phase was indistinguishable from the calcium suppression induced by A23187 administration. Since Hardy type mutation on amyIoid precursor protein gene is found in the OS-1 family, the observed abnormalities in calcium response might be related with pathological processing of amyloid precursor protein in AD. The reported abnormal calcium response, which is observed most obviously in fibroblasts in S phase, may indicate participation of the cell-cycle-dependent process in the pathology of AD.