Yasuhisa Ano

Spherical lactic acid bacteria activate plasmacytoid dendritic cells immunomodulatory function via TLR9-dependent crosstalk with myeloid dendritic cells.

Plasmacytoid dendritic cells (pDC) are a specialized sensor of viral and bacterial nucleic acids and a major producer of IFN-α that promotes host defense by priming both innate and acquired immune responses. Although synthetic Toll-like receptor (TLR) ligands, pathogenic bacteria and viruses activate pDC, there is limited investigation of non-pathogenic microbiota that are in wide industrial dietary use, such as lactic acid bacteria (LAB). In this study, we screened for LAB strains, which induce pDC activation and IFN-α production using murine bone marrow (BM)-derived Flt-3L induced dendritic cell culture. Microbial strains with such activity on pDC were absent in a diversity of bacillary strains, but were observed in certain spherical species (Lactococcus, Leuconostoc, Streptococcus and Pediococcus), which was correlated with their capacity for uptake by pDC. Detailed study of Lactococcus lactis subsp. lactis JCM5805 and JCM20101 revealed that the major type I and type III interferons were induced (IFN-α, -β, and λ). IFN-α induction was TLR9 and MyD88-dependent; a slight impairment was also observed in TLR4-/- cells. While these responses occurred with purified pDC, IFN-α production was synergistic upon co-culture with myeloid dendritic cells (mDC), an interaction that required direct mDC-pDC contact. L. lactis strains also stimulated expression of immunoregulatory receptors on pDC (ICOS-L and PD-L1), and accordingly augmented pDC induction of CD4+CD25+FoxP3+ Treg compared to the Lactobacillus strain. Oral administration of L. lactis JCM5805 induced significant activation of pDC resident in the intestinal draining mesenteric lymph nodes, but not in a remote lymphoid site (spleen). Taken together, certain non-pathogenic spherical LAB in wide dietary use has potent and diverse immunomodulatory effects on pDC potentially relevant to anti-viral immunity and chronic inflammatory disease.

Oxidative damage to neurons caused by the induction of microglial NADPH oxidase in encephalomyocarditis virus infection.

Reactive oxygen species (ROS) play an important role in diverse vital functions including host defense via anti-viral and anti-bacterial effects, but ROS also lead to peroxynitrite and hydroxyl radical production, which are powerful mediators of brain injury in brain inflammation. It is known that NADPH oxidases (NOX) are the major source of ROS. In the present study, NOX2 expression and distribution were examined after intracranial encephalomyocarditis virus B variant (EMCV-B) infection, which causes encephalitis. The reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunohistochemistry showed that the expression and distribution of NOX2 were significantly up-regulated after EMCV-B infection in microglial cells, which invaded into the surrounding regions where neurons were subjected to oxidative stress. These findings suggest that the oxidative stress generated by NOX2 in activated microglial cells damages neurons and that this is an important process in the pathogenesis of EMCV-B infection.

Uptake and Dynamics of Infectious Prion Protein in the Intestine

Transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation of a protease-resistant abnormal isoform of the prion protein (PrPSc), which is converted from the cellular isoform of the prion protein (PrPC). In the oral transmission of prion protein, PrPSc can invade a host body through the intestinal tract. There is only limited information available on how the infectious agent passes through one or several biological barriers before it can finally reach the brain. After oral administration, PrPSc withstands the digestive process and may be incorporated by microfold (M) cells or villous columnar epithelial cells in the intestine. After entry into the intestinal epithelium, PrPSc accumulates and is amplified in follicular dendritic cells (FDCs) within Peyers patches and other isolated lymphoid follicles possibly by an interaction with dendritic cells or macrophages. Following accumulation in gut-associated lymphoid tissues, PrPSc is thought to move to the enteric nervous systems (ENS) by an interaction with FDCs or dendritic cells. As a result of neuroinvasion into the ENS, PrPSc spreads to the central nervous system. In addition, an epidemiological study suggested that most bovine spongiform encephalopathy cases had been exposed to the agent in the first 6 months of life. Developments of the intestinal defense and immune system may be involved in the susceptibility to infection.

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies.

AIMS Insulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice. MAIN METHODS To localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2(+/+) mice. IA-2(-/-) mice served as a negative control. KEY FINDINGS Western blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells. SIGNIFICANCE The IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.

Cellular prion protein prevents brain damage after encephalomyocarditis virus infection in mice

Cellular prion protein (PrPC), a cell-surface glycoprotein normally associated with neurons, is also expressed in other cell types such as glia and lymphocytes. To further elucidate these roles of PrPC, wild-type prion protein gene (Prnp+/+) mice and Prnp-deficient (Prnp−/−) mice were infected with encephalomyocarditis virus B variant (EMCV-B) via an intracranial route. EMCV-B causes encephalitis and apoptotic cell death in vivo. Histopathological studies revealed that Prnp+/+ mice infected with 600 pfu of EMCV-B showed more severe infiltration of inflammatory cells, accompanied by higher activation of microglia cells around the hippocampus, than Prnp−/− mice; viz., no differences in the brain virus titer between these two lines of mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP, nick end-labeling (TUNEL) staining of the brain specimens revealed that the CA1 hippocampal pyramidal cells showed a larger number of apoptotic neurons in Prnp−/− than Prnp+/+ mice. Based on all these findings, PrPC may play certain roles in the induction of inflammation and inhibition of apoptosis in vivo.

Fundamentals of prions and their inactivation (review).

Prion is an infectious particle composed of an abnormal isoform of the prion protein (PrPSc) and causes prion diseases such as bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) and scrapie. Host cells express cellular prion protein (PrPC), which plays roles in normal functions such as anti-oxidative stress. PrPSc is derived from PrPC and produced by conformational conversion. Prion is notorious as a resistant pathogen, being difficult to inactivate with conventional sterilization procedures. Therefore, to prevent prion-caused iatrogenic diseases, the use of appropriate procedures to inactivate prions is important. For examples, alcohol treatment, autoclave (121˚C, 20 min) and γ-ray irradiation, which are used for disinfection, antisepsis or sterilization of viruses and bacteria, are not effective against prion. This is a fundamental review of prions and methods of their inactivation.

Preventive Effects of a Fermented Dairy Product against Alzheimer’s Disease and Identification of a Novel Oleamide with Enhanced Microglial Phagocytosis and Anti-Inflammatory Activity

Despite the ever-increasing number of patients with dementia worldwide, fundamental therapeutic approaches to this condition have not been established. Epidemiological studies suggest that intake of fermented dairy products prevents cognitive decline in the elderly. However, the active compounds responsible for the effect remain to be elucidated. The present study aims to elucidate the preventive effects of dairy products on Alzheimer’s disease and to identify the responsible component. Here, in a mouse model of Alzheimer’s disease (5xFAD), intake of a dairy product fermented with Penicillium candidum had preventive effects on the disease by reducing the accumulation of amyloid β (Aβ) and hippocampal inflammation (TNF-α and MIP-1α production), and enhancing hippocampal neurotrophic factors (BDNF and GDNF). A search for preventive substances in the fermented dairy product identified oleamide as a novel dual-active component that enhanced microglial Aβ phagocytosis and anti-inflammatory activity towards LPS stimulation in vitro and in vivo. During the fermentation, oleamide was synthesized from oleic acid, which is an abundant component of general dairy products owing to lipase enzymatic amidation. The present study has demonstrated the preventive effect of dairy products on Alzheimer’s disease, which was previously reported only epidemiologically. Moreover, oleamide has been identified as an active component of dairy products that is considered to reduce Aβ accumulation via enhanced microglial phagocytosis, and to suppress microglial inflammation after Aβ deposition. Because fermented dairy products such as camembert cheese are easy to ingest safely as a daily meal, their consumption might represent a preventive strategy for dementia.

Iso-α-acids, Bitter Components of Beer, Prevent Inflammation and Cognitive Decline Induced in a Mouse Model of Alzheimer's Disease

Alongside the rapid growth in aging populations worldwide, prevention and therapy for age-related memory decline and dementia are in great demand to maintain a long, healthy life. Here we found that iso-α-acids, hop-derived bitter compounds in beer, enhance microglial phagocytosis and suppress inflammation via activation of the peroxisome proliferator-activated receptor γ. In normal mice, oral administration of iso-α-acids led to a significant increase both in CD11b and CD206 double-positive anti-inflammatory type microglia (p < 0.05) and in microglial phagocytosis in the brain. In Alzheimers model 5xFAD mice, oral administration of iso-α-acids resulted in a 21% reduction in amyloid β in the cerebral cortex as observed by immunohistochemical analysis, a significant reduction in inflammatory cytokines such as IL-1β and chemokines including macrophage inflammatory protein-1α in the cerebral cortex (p < 0.05) and a significant improvement in a novel object recognition test (p < 0.05), as compared with control-fed 5xFAD mice. The differences in iso-α-acid-fed mice were due to the induction of microglia to an anti-inflammatory phenotype. The present study is the first to report that amyloid β deposition and inflammation are suppressed in a mouse model of Alzheimers disease by a single component, iso-α-acids, via the regulation of microglial activation. The suppression of neuroinflammation and improvement in cognitive function suggests that iso-α-acids contained in beer may be useful for the prevention of dementia.

Structure of the Prion Protein and Its Gene: An Analysis Using Bioinformatics and Computer Simulation

Prion protein (PrP) gene encodes cellular PrP (PrPC), a glycosylphosphatidylinositol (GPI)-anchored cell membrane protein indispensable for infections of prion, which causes Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. Although PrPC is known to be converted into an abnormal isoform (PrPSc) upon prion infection and play an important role in prion diseases, the mechanisms involved remain unclear, partly due to the insolubility of PrPSc, which prevents experimental biochemical and biophysical analyses. Recently, with improvements in computer power and methods, computer analyses have been contributing more to prion studies. A comparison of PrP gene sequences revealed mutations and polymorphisms in the open reading frame (ORF) of the human PrP gene related to prion diseases. In contrast, little mutations or polymorphisms related to susceptibility to BSE were found in the ORF of the bovine PrP gene, though relationships between insertion/deletion (Ins/Del) polymorphisms of the PrP gene promoter and susceptibility to BSE have been found. Our results have shown that the specific protein 1 (Sp1) plays important role in the activity of PrP gene promoter, which is influenced by polymorphisms in the Sp1 binding sites. The potential structural dynamics of PrP have been simulated by computational methods such as molecular dynamics (MD) and quantum mechanics (QM). The proposed mechanisms of conversion have revealed new insights in prion diseases. In this review, we will introduce the gene structure, polymorphisms, and potential structural dynamics of PrP revealed by basic and advanced computational analyses. The possible contribution of these methods to elucidation of the pathogenicity of prion diseases and functions of PrPC is discussed.

Incorporation of β-amyloid protein through the bovine ileal epithelium before and after weaning: Model for orally transmitted amyloidoses

To determine the mechanism of bovine intestinal incorporation of the pathogen, and the pathogenesis of prion protein in the early stage, cows suckling and weaning were orally given a fusion protein of Aβ‐EGFP. Aβ‐EGFP was incorporated through the villous columnar epithelial cells and accumulated in crypt patches in the ileum of suckling cows. The sites of the uptake and accumulation of Aβ‐EGFP are very close to the peripheral nervous system; however, such uptake of Aβ‐EGFP was not observed in 6‐month‐old post‐weaning cows. The present study, therefore, suggests that the weaning period is very important for the risk of transmission.