In Japan, neuropsychological symptoms after human papillomavirus (HPV) vaccinations were publicized as "adverse effects," leading to vaccine hesitancy. Anti-vaccine activists claimed that adjuvants in HPV vaccines could cause an immune-mediated neurological disease. Adjuvants in the bivalent HPV vaccine (2vHPV) and quadrivalent HPV vaccine (4vHPV) are AS04 [composed of aluminum (Al) hydroxide (AH) and monophosphoryl lipid A (MPL)] and Al hydroxyphosphate sulfate (AHS), respectively. We determined whether HPV vaccinations in mice could reproduce alleged immunopathology. We injected mice intramuscularly with 2vHPV, 4vHPV, two hepatitis B virus vaccines containing AH or AHS, or a varicella-zoster virus vaccine (vVZV) containing an adjuvant AS01 (comprising MPL and QS-21). Histologically, 12 weeks after vaccinations, all four Al-containing vaccine groups had Al-laden macrophage accumulation at the injected muscle; no groups had abnormalities in any other organs, including the brain, heart, liver, and kidney. Immunologically, although the four Al-containing vaccine groups had continuously increased levels of several cytokines, including interferon (IFN)-β, cytokine profiles were not associated with muscle pathology. No groups exhibited any clinical signs, except for the vVZV group, which lost body weight temporarily following each injection. Weight loss in the vVZV group was associated with increased levels of cytokines, including interleukin (IL)-18. Experiments using IL-18 receptor-deficient mice and AS01 injection alone demonstrated that IL-18 and AS01 contributed to weight loss. Since 2vHPV containing AS04 (AH and MPL) did not induce weight loss, QS-21, but not MPL, in AS01 seemed responsible for weight loss, demonstrating the safety of MPL.

Upper airway samples, including nasal lavage fluid (NLF) and nasal swabs, are useful in detecting pathogens, including viruses and bacteria, which can cause respiratory diseases. NLF has been used to examine cellular and humoral components in the respiratory system, for example, in evaluating the induction of immunoglobulin (Ig) A following mucosal vaccinations. In experimental rodents, the NLF samples can be collected by either the trans-pharyngeal or trans-tracheal route. Although the trans-pharyngeal route has been reported to be more efficient in collecting the NLF samples than the trans-tracheal route, the NLF samples collected by the trans-pharyngeal route were often contaminated with blood, affecting the levels of cellular and humoral contents in the original NLF samples. Thus, this study aimed to establish a novel NLF collection method in experimental mice to minimize blood contamination. Briefly, before the lower jaw of the mouse was separated, cotton balls were placed in the mouth to absorb blood. When the bleeding was stopped, the NLF samples were collected by inserting a micropipette into the choana and flushing with 200 µL of phosphate-buffered saline twice (final volume: 400 µL/mouse). To detect blood contamination, a simple and sensitive forensic luminol test that detects hemoglobin was used. The luminol test demonstrated that blood contamination was detectable in the NLF samples harvested by the conventional method (without cotton balls), but not those harvested by the novel method (with cotton balls). Due to the contamination of blood, the total IgA and IgG concentrations in the NLF samples were higher in the conventional method than in the novel method; blood has been known to contain much higher levels of IgA and IgG than NLF. Therefore, this unique method can be used as a simple and sensitive method to collect NLF samples from experimental mice to prevent blood contamination.

Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain–Barré syndrome. On the other hand, the role in multiple sclerosis (MS), inflammatory demyelinating disease in the central nervous system (CNS), is largely unknown, although the presence of anti-glycolipid antibodies was reported to differ among MS patients with relapsing-remitting (RR), primary progressive (PP), and secondary progressive (SP) disease courses. We investigated whether the induction of anti-glycolipid antibodies could differ among experimental MS models with distinct clinical courses, depending on induction methods. Using three mouse strains, SJL/J, C57BL/6, and A.SW mice, we induced five distinct experimental autoimmune encephalomyelitis (EAE) models with myelin oligodendrocyte glycoprotein (MOG)35–55, MOG92–106, or myelin proteolipid protein (PLP)139–151, with or without an additional adjuvant curdlan injection. We also induced a viral model of MS, using Theiler’s murine encephalomyelitis virus (TMEV). Each MS model had an RR, SP, PP, hyperacute, or chronic clinical course. Using the sera from the MS models, we quantified antibodies against 11 glycolipids: GM1, GM2, GM3, GM4, GD3, galactocerebroside, GD1a, GD1b, GT1b, GQ1b, and sulfatide. Among the MS models, we detected significant increases in four anti-glycolipid antibodies, GM1, GM3, GM4, and sulfatide, in PLP139–151-induced EAE with an RR disease course. We also tested cellular immune responses to the glycolipids and found CD1d-independent lymphoproliferative responses only to sulfatide with decreased interleukin (IL)-10 production. Although these results implied that anti-glycolipid antibodies might play a role in remissions or relapses in RR-EAE, their functional roles need to be determined by mechanistic experiments, such as injections of monoclonal anti-glycolipid antibodies.

Abstract Helicobacter pylori (HP) is a Gram-negative bacterium that colonizes the human stomach chronically. Colonization of HP in the gastric mucosa not only causes gastrointestinal diseases, but also is associated with extra-gastric diseases, such as idiopathic thrombocytopenic purpura and neurological diseases. Among neurological diseases, epidemiological studies have shown that HP infection increases the prevalence of Alzheimer’s disease (AD) and Parkinson’s disease (PD). Since HP does not invade the central nervous system (CNS), it has been considered that systemic immunological changes induced by HP infection may play pathogenic roles in AD and PD. Here, we investigated the effects of HP infection on the CNS in vivo and in vitro. In the CNS, chronically HP-infected mice had microglial activation without HP colonization, although systemic immunological changes were not observed. This led us to explore the possibility that HP-derived outer membrane vesicles (HP-OMVs) could cause neuroinflammation. OMVs are small, spherical bilayer vesicles (20–500 nm) released into the extracellular space from the outer membrane of Gram-negative bacteria; OMVs contain lipopolysaccharide, proteins, peptidoglycan, DNA, and RNA. OMVs have also been shown to activate both innate and acquired immune cells in vitro, and to disrupt the tight junctions of the gastric epithelium (“leaky gut”) as well as cross the blood-brain barrier in vivo. Thus, in theory, OMVs can activate immune responses in the remote organs, including the lymphoid organs and CNS, if only OMVs enter the systemic circulation. From the exosome fraction of sera from HP-infected mice, we detected HP-specific DNA, suggesting the presence of HP-OMVs. We also found that microglia incubated with HP-OMVs in vitro increased the cell proliferation, inflammatory cytokine production, and migration. On the other hand, HP-OMVs suppressed the cell proliferation of neuroblastoma in vitro. Lastly, we found that AD model mice infected with HP had amyloid plaques adjacent to activated microglia and astrocytes in vivo. Based on the literature review and our experimental data, we propose our working hypothesis that OMVs produced in chronic HP infection in the gut induce neuroinflammation in the CNS, explaining the higher prevalence of AD in HP-infected people.

Abstract Autoimmune pancreatitis (AIP) and IgG4-related disease (IgG4-RD) are new disease entities characterized by enhanced IgG4 antibody responses and involvement of multiple organs, including the pancreas and salivary glands. Although the immunopathogenesis of AIP and IgG4-RD is poorly understood, we previously reported that intestinal dysbiosis mediates experimental AIP through the activation of IFN-α- and IL-33-producing plasmacytoid dendritic cells (pDCs). Because intestinal dysbiosis is linked to intestinal barrier dysfunction, we explored whether the latter affects the development of AIP and autoimmune sialadenitis in MRL/MpJ mice treated with repeated injections of polyinosinic–polycytidylic acid [poly (I:C)]. Epithelial barrier disruption was induced by the administration of dextran sodium sulfate (DSS) in the drinking water. Mice co-treated with poly (I:C) and DSS, but not those treated with either agent alone, developed severe AIP, but not autoimmune sialadenitis, which was accompanied by the increased accumulation of IFN-α- and IL-33-producing pDCs. Sequencing of 16S ribosomal RNA revealed that Staphylococcus sciuri translocation from the gut to the pancreas was preferentially observed in mice with severe AIP co-treated with DSS and poly (I:C). The degree of experimental AIP, but not of autoimmune sialadenitis, was greater in germ-free mice mono-colonized with S. sciuri and treated with poly (I:C) than in germ-free mice treated with poly (I:C) alone, which was accompanied by the increased accumulation of IFN-α- and IL-33-producing pDCs. Taken together, these data suggest that intestinal barrier dysfunction exacerbates AIP through the activation of pDCs and translocation of S. sciuri into the pancreas.

The COVID-19 pandemic has led people to wear face masks daily in public. Although the effectiveness of face masks against viral transmission has been extensively studied, there have been few reports on potential hygiene issues due to bacteria and fungi attached to the face masks. We aimed to (1) quantify and identify the bacteria and fungi attaching to the masks, and (2) investigate whether the mask-attached microbes could be associated with the types and usage of the masks and individual lifestyles. We surveyed 109 volunteers on their mask usage and lifestyles, and cultured bacteria and fungi from either the face-side or outer-side of their masks. The bacterial colony numbers were greater on the face-side than the outer-side; the fungal colony numbers were fewer on the face-side than the outer-side. A longer mask usage significantly increased the fungal colony numbers but not the bacterial colony numbers. Although most identified microbes were non-pathogenic in humans; Staphylococcus epidermidis, Staphylococcus aureus, and Cladosporium, we found several pathogenic microbes; Bacillus cereus, Staphylococcus saprophyticus, Aspergillus, and Microsporum. We also found no associations of mask-attached microbes with the transportation methods or gargling. We propose that immunocompromised people should avoid repeated use of masks to prevent microbial infection.

Abstract Liver damage affects the prognosis of patients with erythropoietic protoporphyria (EPP). However, there is no radical cure for EPP patients with severe liver damage. This study aims to investigate the effectiveness of phlebotomy in patients with severe liver damage. We examined seven patients diagnosed with EPP and liver damage between 2010 and 2020. Of the 7 cases, phlebotomy was performed in 3 cases with severe hepatic disorder, and the improvement effect of hepatic disorder was observed in all cases. In addition, as an additional study, we also investigated the mechanism by which liver damage becomes more severe. Liver biopsy samples were stained with hematoxylin and eosin and immunohistochemistry was used to examine the expression of adenosine triphosphate-binding transporter G2 (ABCG2). Liver biopsies were performed in 3 of 7 patients with EPP. Of these three patients, ABCG2 expression was low in two patients, especially in the protoporphyrin (PP) deposition area. Two patients with reduced ABCG2 expression subsequently developed severe liver damage. However, the causal relationship between the decreased expression of ABCG2 and the exacerbation of liver damage has not been directly proved, and further investigation is required in the future. This study demonstrated the effectiveness of phlebotomy in EPP patients with severe liver damage.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) are widely used to treat advanced metastatic cancers. Neutralisation of PD-1 or CTLA-4 by ICIs results in immune-related adverse events (irAEs). The clinicopathological features of twelve patients with hepatic irAEs were evaluated and compared to those of ten patients with autoimmune hepatitis (AIH) or graft-versus-host disease (GVHD). No significant difference was seen in serum levels of transaminases, whereas serum levels of IgG and anti-nuclear antibody were higher in patients with AIH than in those with GVHD or hepatic irAEs. Inflammation was limited to the liver lobes in patients with GVHD or hepatic irAEs, whereas patients with AIH exhibited both portal and lobular inflammation. Immunohistochemical analyses revealed a predominant infiltration of CD8⁺ T cells and defective accumulation of regulatory T cells (Tregs) expressing forkhead box p3 (FOXP3) in the lobular areas of patients with hepatic irAEs and GVHD. In contrast, periportal lesions of patients with AIH were characterised by an infiltration of CD4⁺ T cells, CD8⁺ T cells, CD20⁺ B cells, and FOXP3⁺ Tregs. Overall, the activation of CD8⁺ T cells in the absence of activation of Tregs potentially underlies the immunopathogenesis of hepatic irAEs.

Non-steroidal anti-inflammatory drugs (NSAIDs) induce ulcers in the gastrointestinal tract, including the stomach and small intestine. NSAID-induced gastric ulcers can be prevented by taking acid-neutralizing/inhibitory drugs and cytoprotective agents. In contrast, there are no medicines to control NSAID-induced small intestinal ulcers, which are accompanied by a mucosal invasion of bacteria and subsequent activation of immune cells. Galectin-3 (Gal3), an endogenous lectin, has anti-microbial and pro-inflammatory functions. In the small intestine, since Gal3 is highly expressed in epithelial cells constitutively and macrophages inducibly, the Gal3 level can affect microbiota composition and macrophage activation. We hypothesized that the modulation of Gal3 expression could be beneficial in NSAID-induced intestinal ulcers. Using Gal3 knockout (Gal3KO) mice, we determined whether Gal3 could be a therapeutic target in NSAID-induced intestinal ulcers. Following the administration of indomethacin, an NSAID, we found that small intestinal ulcers were less severe in Gal3KO mice than in wild-type (WT) mice. We also found that the composition of intestinal microbiota was different between WT and Gal3KO mice and that bactericidal antibiotic polymyxin B treatment significantly suppressed NSAID-induced ulcers. Furthermore, clodronate, a macrophage modulator, attenuated NSAID-induced ulcers. Therefore, Gal3 could be an exacerbating factor in NSAID-induced intestinal ulcers by affecting the intestinal microbiota population and macrophage activity. Inhibition of Gal3 may be a therapeutic strategy in NSAID-induced intestinal ulcers. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03832946.

Virus infections have been associated with acute and chronic inflammatory central nervous system (CNS) diseases, e.g., acute flaccid myelitis (AFM) and multiple sclerosis (MS), where animal models support the pathogenic roles of viruses. In the spinal cord, Theiler's murine encephalomyelitis virus (TMEV) induces an AFM-like disease with gray matter inflammation during the acute phase, 1 week post infection (p.i.), and an MS-like disease with white matter inflammation during the chronic phase, 1 month p.i. Although gut microbiota has been proposed to affect immune responses contributing to pathological conditions in remote organs, including the brain pathophysiology, its precise role in neuroinflammatory diseases is unclear. We infected SJL/J mice with TMEV; harvested feces and spinal cords on days 4 (before onset), 7 (acute phase), and 35 (chronic phase) p.i.; and examined fecal microbiota by 16S rRNA sequencing and CNS transcriptome by RNA sequencing. Although TMEV infection neither decreased microbial diversity nor changed overall microbiome patterns, it increased abundance of individual bacterial genera Marvinbryantia on days 7 and 35 p.i. and Coprococcus on day 35 p.i., whose pattern-matching with CNS transcriptome showed strong correlations: Marvinbryantia with eight T-cell receptor (TCR) genes on day 7 and with seven immunoglobulin (Ig) genes on day 35 p.i.; and Coprococcus with gene expressions of not only TCRs and IgG/IgA, but also major histocompatibility complex (MHC) and complements. The high gene expression of IgA, a component of mucosal immunity, in the CNS was unexpected. However, we observed substantial IgA positive cells and deposition in the CNS, as well as a strong correlation between CNS IgA gene expression and serum anti-TMEV IgA titers. Here, changes in a small number of distinct gut bacteria, but not overall gut microbiota, could affect acute and chronic immune responses, causing AFM- and MS-like lesions in the CNS. Alternatively, activated immune responses would alter the composition of gut microbiota.

Autoimmune pancreatitis (AIP) is a pancreatic manifestation of a newly proposed disease entity, IgG4-related disease (IgG4-RD), characterized by enhanced IgG4 antibody responses and involvement of multiple organs. We have previously reported that innate immune activation contributes to the development of AIP and IgG4-RD, as these diseases are characterized by the production of IFN-α and IL-33 by plasmacytoid dendritic cells (pDCs) that mediate chronic fibroinflammatory responses. In this study, we investigated the roles played by innate immunity against intestinal microflora in experimental AIP induced in MRL/MpJ mice by repeated administrations of 100 µg of polyinosinic-polycytidylic acid [poly (I:C)]. Bowel sterilization with a broad spectrum of antibiotics inhibited pancreatic accumulation of pDCs producing IFN-α and IL-33, and thereby suppressed the development of AIP. Mice treated with 10 µg of poly (I:C) developed severe AIP equivalent to that induced by 100 µg of poly (I:C) upon co-housing with mice treated with 100 µg of poly (I:C). Fecal microbiota transplantation (FMT) from donor mice treated with 100 µg of poly (I:C) led to the development of severe AIP in the recipient mice upon injection with 10 µg of poly (I:C). Induction of severe AIP in mice with 10 µg of poly (I:C) was associated with pancreatic accumulation of pDCs producing IFN-α and IL-33 in the co-housing and FMT experiments. These data collectively suggest that innate immune responses against intestinal microflora are involved in the development of experimental AIP, and that intestinal dysbiosis increases sensitivity to experimental AIP via activation of pDCs.

Previously, we have established two distinct progressive multiple sclerosis (MS) models by induction of experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) in two mouse strains. A.SW mice develop ataxia with antibody deposition, but no T cell infiltration, in the central nervous system (CNS), while SJL/J mice develop paralysis with CNS T cell infiltration. In this study, we determined biomarkers contributing to the homogeneity and heterogeneity of two models. Using the CNS and spleen microarray transcriptome and cytokine data, we conducted computational analyses. We identified up-regulation of immune-related genes, including immunoglobulins, in the CNS of both models. Pro-inflammatory cytokines, interferon (IFN)-γ and interleukin (IL)-17, were associated with the disease progression in SJL/J mice, while the expression of both cytokines was detected only at the EAE onset in A.SW mice. Principal component analysis (PCA) of CNS transcriptome data demonstrated that down-regulation of prolactin may reflect disease progression. Pattern matching analysis of spleen transcriptome with CNS PCA identified 333 splenic surrogate markers, including Stfa2l1, which reflected the changes in the CNS. Among them, we found that two genes (PER1/MIR6883 and FKBP5) and one gene (SLC16A1/MCT1) were also significantly up-regulated and down-regulated, respectively, in human MS peripheral blood, using data mining.

Heat shock protein 27 (HSP27) protects cells under stress. Here, we demonstrate that HSP27 also promotes cell cycle progression of MRC-5 human lung fibroblast cells. Serum starvation for 24 h induced G1 arrest in these cells, and upon serum refeeding, the cells initiated cell cycle progression accompanied by an increase in HSP27 protein levels. HSP27 levels peaked at 12 h, and transcriptional up-regulation of six G2/M-related genes (CCNA2, CCNB1, CCNB2, CDC25C, CDCA3, and CDK1) peaked at 24-48 h. siRNA-mediated HSP27 silencing in proliferating MRC-5 cells induced G2 arrest coinciding with down-regulation of these six genes. Of note, the promoters of all of these genes have the cell cycle-dependent element and/or the cell cycle gene-homology region. These promoter regions are known to be bound by the E2F family proteins (E2F-1 to E2F-8) and retinoblastoma (RB) family proteins (RB1, p107, and p130), among which E2F-4 and p130 were strongly up-regulated in HSP27-knockdown cells. E2F-4 or p130 knockdown concomitant with the HSP27 knockdown rescued MRC-5 cells from G2 arrest and up-regulated the six cell cycle genes. Moreover, we observed cellular senescence in MRC-5 cells on day 3 after the HSP27 knockdown, as evidenced by increased senescence-associated β-gal activity and up-regulated inflammatory cytokines. The cellular senescence was also suppressed by the concomitant knockdown of E2F-4/HSP27 or p130/HSP27. Our findings indicate that HSP27 promotes cell cycle progression of MRC-5 cells by suppressing expression of the transcriptional repressors E2F-4 and p130.

Fecal occult blood (FOB) is a sign of gastrointestinal diseases, such as intestinal ulcers and colorectal cancer. In experimental animal studies, there is no standard method to detect FOB. Here, we present a simple protocol to detect FOB in mice, using the Luminol Reaction Experiment Kit® that was originally designed to detect bloodstains at a crime scene in criminal forensics. To obtain positive control bloody feces, we used an indomethacin-induced intestinal ulcer model in mice. By mixing small pieces of feces with a luminol solution, the fecal solution emitted visible blue-white chemiluminescence in dark field when feces contained hemoglobin. We also established a method for semi-quantification of hemoglobin content in the fecal solution, using a luminometer. This method is simple, quick, economical and semi-quantitative, allowing researchers to detect FOB in experimental mice.

Purpose: Staphylococcus aureus is a common cause of corneal ulceration, and staphylokinase (SAK) produced by this bacterium is a plasminogen activator. To investigate the pathogenesis of corneal ulceration induced by S. aureus, we examined the effects of bacterial culture broth and SAK on collagen degradation in a culture model in which human corneal fibroblasts are embedded in a collagen gel. Methods: Corneal fibroblasts embedded in collagen were exposed to S. aureus culture broth or SAK. Collagen degradation was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Expression of pro-matrix metalloproteinase-1 (pro-MMP-1) was detected by immunoblot analysis as well as reverse transcription and real-time polymerase chain reaction analysis. Results: Both S. aureus culture broth and SAK markedly increased collagen degradation in the presence of corneal fibroblasts and plasminogen. This effect of the culture broth was dependent on cell number to a greater extent than was that of SAK. Whereas the culture broth also increased the expression of pro-MMP-1 in corneal fibroblasts at both mRNA and protein levels, SAK did not. Conclusions: Our results suggest that S. aureus may promote collagen degradation both by upregulating pro-MMP1 expression in corneal fibroblasts, with pro-MMP-1 then being converted to active MMP-1 by plasmin, and by directing plasmin activity toward collagen in a SAK-dependent manner.

Alteration of microbiota has been associated with intestinal, inflammatory, and neurological diseases. Abundance of "good bacteria" such as Bifidobacterium, or their products have been generally believed to be beneficial for any diseases, while "bad bacteria" such as pathogenic Helicobacter pylori are assumed to be always detrimental for hosts. However, this is not the case when we compare and contrast the association of the gut microbiota with two neurological diseases, multiple sclerosis (MS) and Alzheimer's disease (AD). Following H. pylori infection, pro-inflammatory T helper (Th)1 and Th17 immune response are initially induced to eradicate bacteria. However, H. pylori evades the host immune response by inducing Th2 cells and regulatory T cells (Tregs) that produce anti-inflammatory interleukin (IL)-10. Suppression of anti-bacterial Th1/Th17 cells by Tregs may enhance gastric H. pylori propagation, followed by a cascade reaction involving vitamin B12 and folic acid malabsorption, plasma homocysteine elevation, and reactive oxygen species induction. This can damage the blood-brain barrier (BBB), leading to accumulation of amyloid-β in the brain, a hallmark of AD. On the other hand, this suppression of pro-inflammatory Th1/Th17 responses to H. pylori has protective effects on the hosts, since it prevents uncontrolled gastritis as well as suppresses the induction of encephalitogenic Th1/Th17 cells, which can mediate neuroinflammation in MS. The above scenario may explain why chronic H. pylori infection is positively associated with AD, while it is negatively associated with MS. Lastly, we list "10 pitfalls of microbiota studies", which will be useful for evaluating and designing clinical and experimental microbiota studies.

Theiler's murine encephalomyelitis virus (TMEV) infection has been used as a viral model for multiple sclerosis (MS), as TMEV can induce chronic inflammatory demyelinating lesions with viral persistence in the spinal cord of SJL/J mice. In contrast, when C57BL/6 mice are infected with TMEV, the mice can clear the virus from the central nervous system (CNS), without viral persistence or demyelination, but develop seizures and hippocampal sclerosis, which has been used as a viral model for seizures/epilepsy. In the two TMEV-induced CNS disease models, not only viral infection, but also immune responses contribute to the pathogenesis. Interestingly, acquired immunity plays an effector role in the MS model, whereas innate immunity appears to contribute to the development of seizures. Recently, we have established the third TMEV-induced disease model, a mouse model for viral myocarditis, using C3H mice. TMEV-induced myocarditis is a triphasic disease, which mimics human myocarditis; phase I, mediated by viral replication in the heart and innate immunity; phase II, mediated by acquired immunity; and phase III, resulted from cardiac fibrosis. The genetic susceptibility to the aforementioned three models (MS, seizures and myocarditis) differs among mouse strains. We have compared and contrasted the three models induced by one single pathogen, TMEV, particularly in regard to the roles of T helper cells and natural killer T cells, which will give an insight into how interactions between the immune system and the host's genetic background determine the tissue tropism of virus and the development of virus-induced organ-specific immunopathology.

Zika virus (ZIKV) is an enveloped, positive-sense, single-stranded RNA virus that belongs to the genus Flavivirus, family Flaviviridae, which includes many human and animal pathogens, such as dengue virus (DENV), West Nile virus, and Japanese encephalitis virus. In the original as well as subsequent experimental and clinical reports, ZIKV seems to have moderate neurotropism (in animal models) and neurovirulence (in human fetuses), but no neuroinvasiveness (in human adults). Intrauterine ZIKV infection (viral pathology) has been linked to an increased incidence of microcephaly, while increased Guillain-Barré syndrome (GBS) following ZIKV infection is likely immune-mediated (immunopathology). Clinically, in ZIKV infection, antibodies against other flaviviruses, such as DENV, have been detected; these antibodies can cross-react with ZIKV without ZIKV neutralization. In theory, such non-neutralizing antibodies are generated at the expense of decreased production of neutralizing antibodies ("antigenic sin"), leading to poor viral clearance, while the non-neutralizing antibodies can also enhance viral replication in Fc receptor (FcR)-bearing cells via antibody-dependent enhancement (ADE). Here, we propose three potential roles of the antibody-mediated pathogenesis of ZIKV infection: 1) cross-reactive antibodies that recognize ZIKV and neural antigens cause GBS; 2) ZIKV-antibody complex is transported transplacentally via neonatal FcR (FcRn), resulting in fetal infection; and 3) ZIKV-antibody complex is taken up at peripheral nerve endings and transported to neurons in the central nervous system (CNS), by which the virus can enter the CNS without crossing the blood-brain barrier.

Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.

In vivo study アルツハイマーモデル（AD）マウスと野生型(WT)マウスにヘリコバクター・ピロリ菌（HP）を長期間（10ヶ月）感染させ、新奇物体探索テストにより認知行動/運動量の解析後に血液、脳と胃を回収した。コントロールとして、同齢のHP非感染ADマウスとWTマウスを用いた（AD±HP, WT±HPの4群）。 新奇物体探索テストでの認知行動には4群間で差が見られなかったが、ADマウスでは運動量が亢進しており、特にAD+HPではWT＋HPに比べ有意に増加していた。これは認知症に見られる徘徊行動に類似する状態であるといえる。HP＋マウスでは血中エンドトキシン濃度が有意に増加していた事からHP感染によって消化管がリーキーな状態になっていることが分かった。HP+マウス脳内ではミクログリアの活性化が認められたが、HP-との差は以前に解析した感染5ヶ月後の方が顕著であった。 In vitro study これまでの動物実験解析からHP感染による脳ミクログリアの活性化にはHPが産生する外膜小胞（OMV）が影響している事が考えられたため、ピロリ菌からOMVを回収し、脳の細胞への影響を調べた。WTマウスから脳グリア細胞を回収し、OMVを暴露したところ、IL-6, IL-1bなどの炎症性サイトカインが増加した。脳グリア細胞には主に3種の細胞（ミクログリア、アストロサイト、オリゴデンドロサイト）が混在しているため、それぞれへの影響を調べるため細胞株にOMVを暴露しサイトカインのmRNAを調べた結果、オリゴデンドロサイトは全く反応せず、ミクログリアが最も強く反応した。このことは動物実験結果と一致していた。

Epidemiological studies have reported that the infection rate of H. pylori is significantly higher in Alzheimer's disease. However, there have been few reports that have shown a relationship between them in animal experiments that are not affected by environmental factors. In this study, we found that H. pylori chronic infected mice showed significantly increase of central nervous system inflammation than non-infected mice. Although the mechanism of this increase is not clear at present, we found that the outer membrane vesicles produced by H. pylori may be involved.

The purpose of this study is to clarify the role of Galectin-3 (Gal3) on NSAIDs-induced intestinal ulcer. By using Gal3 knockout mice, we found that Gal-3 is a worsen factor of NSAIDs-induced intestinal ulcer. There are two possible mechanism for this negative effect of Gal3. The first one is a role of Gal3 as danger signal of activated macrophages. This molecule might cause sever inflammation. The another one is changes of intestinal bacterial flora with or without Gal-3. We found obvious differences of small intestinal flora population between wildtype mice and Gal3 mice. Those differences might affect to the ulceration by NSAIDs.

The object of this study is to clarify the epigenetic changes in pulmonary fibrosis. In vivo study using silica-induced mouse fibrosis model revealed that the DNA methylation level was unchanged, but the protein level of DNMT3B, which catalysis DNA methylation, was increased in fibrosis lung. In vitro study also showed the increase of this protein. The increase of DNMT3B protein may play important role in pulmonary fibrosis.

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