Silent progression of brain atrophy in aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder.

OBJECTIVE: To investigate longitudinal brain atrophy in patients with neuromyelitis optica spectrum disorder (NMOSD). METHODS: We investigated the longitudinal brain atrophy rate in patients with aquaporin-4 antibody-positive NMOSD (AQP4+NMOSD) and those with multiple sclerosis (MS) in a retrospective cohort study. Brain volume was calculated with statistical parametric mapping-12. RESULTS: We enrolled 36 patients with AQP4+NMOSD and 60 with MS. Patients with NMOSD were older and had a higher Kurtzke's expanded disability status scale score at baseline MRI compared with those with MS. Disease duration, annual relapse rate and intervals from the last attack and from disease-modifying drugs initiation were not significantly different between the two groups. Lower normalised lesion volume and higher normalised white matter volume were found in patients with NMOSD compared with those with MS at baseline MRI. However, the annualised atrophy rate of normalised brain volume was similar between the NMOSD (median 0.47; IQR 0.75; p=0.49) and MS (median 0.46; IQR 0.84) groups. After adjustment of age and the presence of clinical relapse, no differences of the annualised atrophy rate of normalised brain volume also were found for NMOSD and MS. Patients with AQP4+NMOSD with long cord lesion showed higher annualised atrophy rate of normalised grey matter volume compared with those without long cord lesion. CONCLUSIONS: Silent progression of brain atrophy was present in patients with AQP4+NMOSD, as shown in patients with MS, even in the clinically inactive age-matched cases. Subclinical dying back degeneration may explain the brain atrophy in patients with AQP4 +NMOSD.

Comparative analysis of microRNA expression profiles in the colons of specific pathogen-free mice and germ-free mice.

MicroRNAs play an important role in microbiota-host crosstalk. In this study, we compared microRNA expression in whole colons of specific pathogen-free mice and germ-free mice. Forty-eight microRNAs were differentially expressed by more than 2-fold. Gene ontology analysis of the predicted mRNA targets revealed that the majority of the most significant gene ontology terms were related to GTPases and nerves.

Indole-3-Pyruvic Acid, an Aryl Hydrocarbon Receptor Activator, Suppresses Experimental Colitis in Mice.

Aryl hydrocarbon receptor (AHR) agonists are promising immunomodulators that potentially maintain immune tolerance. In this study, we examined the ability of indole-3-pyruvic acid (IPA), a major precursor of microbiota-derived AHR agonists and a proagonist of AHR, to activate AHR. The anti-inflammatory effects of IPA were also evaluated in a mouse model of colitis in comparison with other aromatic pyruvic acids (phenylpyruvic acid and 4-hydroxyphenylpyruvic acid). Among them, IPA showed the strongest ability to activate AHR in vitro and in vivo, and only IPA improved chronic inflammation in an experimental colitis model. IPA attenuated the expression of genes encoding Th1 cytokines and enhanced Il-10 gene expression in the colon. Oral administration of IPA decreased the frequency of IFN-γ+ IL-10- CD4+ T cells and increased that of IFN-γ- IL-10+ CD4+ T cells in the colon lamina propria in a T cell-mediated colitis model. IPA directly promoted the differentiation of type 1 regulatory T cells in vitro. Furthermore, IPA administration attenuated the ability of dendritic cells (DCs) in the mesenteric lymph nodes (MLN) to induce IFN-γ-producing T cells, increased the frequency of CD103+ CD11b- DCs, and decreased the frequency of CD103- CD11b+ DCs in the MLN. Adoptive transfer of MLN CD103+ CD11b- DCs significantly improved the severity of colon inflammation. Treatment with an AHR antagonist inhibited IPA-induced differentiation of type 1 regulatory T cells and the IPA-induced increase in CD103+ CD11b- DCs and attenuated the anti-inflammatory effect of IPA. These findings suggest that IPA potently prevents chronic inflammation in the colon by activating AHR.

Role of CXC chemokine receptor type 4 as a lactoferrin receptor.

Lactoferrin exerts its biological activities by interacting with receptors on target cells, including LDL receptor-related protein-1 (LRP-1/CD91), intelectin-1 (omentin-1), and Toll-like receptor 4 (TLR4). However, the effects mediated by these receptors are not sufficient to fully explain the many functions of lactoferrin. C-X-C-motif cytokine receptor 4 (CXCR4) is a ubiquitously expressed G-protein coupled receptor for stromal cell-derived factor-1 (SDF-1/CXCL12). Lactoferrin was found to be as capable as SDF-1 in blocking infection by an HIV variant that uses CXCR4 as a co-receptor (X4-tropic HIV), suggesting that lactoferrin interacts with CXCR4. We addressed whether CXCR4 acts as a lactoferrin receptor using HaCaT human keratinocytes and Caco-2 human intestinal cells. We found that bovine lactoferrin interacted with CXCR4-containing lipoparticles, and that this interaction was not antagonized by SDF-1. In addition, activation of Akt in response to lactoferrin was abrogated by AMD3100, a small molecule inhibitor of CXCR4, or by a CXCR4-neutralizing antibody, suggesting that CXCR4 functions as a lactoferrin receptor able to mediate activation of the PI3K-Akt signaling pathway. Lactoferrin stimulation mimicked many aspects of SDF-1-induced CXCR4 activity, including receptor dimerization, tyrosine phosphorylation, and ubiquitination. Cycloheximide chase assays indicated that turnover of CXCR4 was accelerated in response to lactoferrin. These results indicate that CXCR4 is a potent lactoferrin receptor that mediates lactoferrin-induced activation of Akt signaling.

Promoting effect of lactoferrin on barrier function and epithelial differentiation of human keratinocytes.

The purpose of this study was to elucidate the effects of bovine lactoferrin on keratinocyte differentiation and barrier function. Addition of bovine lactoferrin to differentiating HaCaT human keratinocytes led to increased transepithelial electrical resistance (TER), a marker of epithelial barrier function. This elevation was followed by upregulation of two differentiation markers, involucrin and filaggrin. The expression level of sterol regulatory element-binding protein-1 was also enhanced by bovine lactoferrin. The lactoferrin-induced upregulation of involucrin and filaggrin expression were confirmed in normal human epidermal keratinocytes (NHEK). Treatment with SB203580, a p38 mitogen-activated protein kinase (MAPK) α inhibitor, impaired the upregulation of involucrin and filaggrin expression in response to lactoferrin. The elevation of p38 MAPK phosphorylation was further enhanced by lactoferrin in the initial stage of differentiation of HaCaT keratinocytes. The findings suggest that bovine lactoferrin promotes epithelial differentiation by a p38-MAPK-dependent mechanism.

Omics Studies of the Murine Intestinal Ecosystem Exposed to Subchronic and Mild Social Defeat Stress.

The microbiota-gut-brain axis plays an important role in the development of stress-induced mental disorders. We previously established the subchronic and mild social defeat stress (sCSDS) model, a murine experimental model of depression, and investigated the metabolomic profiles of plasma and liver. Here we used omics approaches to identify stress-induced changes in the gastrointestinal tract. Mice exposed to sCSDS for 10 days showed the following changes: (1) elevation of cholic acid and reduction of 5-aminovaleric acid among cecal metabolites; (2) downregulation of genes involved in the immune response in the terminal ileum; (3) a shift in the diversity of the microbiota in cecal contents and feces; and (4) fluctuations in the concentrations of cecal metabolites produced by gut microbiota reflected in plasma and hepatic metabolites. Operational taxonomic units within the family Lachnospiraceae showed an inverse correlation with certain metabolites. The social interaction score correlated with cecal metabolites, IgA, and cecal and fecal microbiota, suggesting that sCSDS suppressed the ileal immune response, altering the balance of microbiota, which together with host cells and host enzymes resulted in a pattern of accumulated metabolites in the intestinal ecosystem distinct from that of control mice.

Prevention of UVB-induced production of the inflammatory mediator in human keratinocytes by lactic acid derivatives generated from aromatic amino acids.

The anti-inflammatory effects of lactic acid derivatives were investigated on ultraviolet B (UVB)-irradiated HaCaT human keratinocytes. A pretreatment with indole-3-lactic acid (ILA) and 4-hydroxyphenyllactic acid (HPLA) inhibited the UVB-induced production of interlekin-6 (IL-6). The inhibitory effect of L-HPLA was equivalent to that of a corresponding racemic mixture (DL-HPLA), suggesting that optical isomerism did not affect the anti-inflammatory activity of HPLA.

Elevated serum levels of bone morphogenetic protein-9 are associated with better outcome in AQP4-IgG seropositive NMOSD.

Lymphatic drainage in the central nervous system is regulated by meningeal lymphatic vasculature, and recurrent neuroinflammation alters lymphatic vessel remodeling. Patients with aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder (AQP4 + NMOSD) were reported to demonstrate worse outcomes compared with patients with anti-myelin oligodendrocyte glycoprotein-associated disorders (MOGAD). This study aimed to investigate the serum cytokines relevant to vascular remodeling after attacks and their prognostic role in patients with AQP4 + NMOSD. This study measured the serum levels of 12 cytokines relevant to vascular remodeling, including bone morphogenetic protein-9 (BMP-9) and leptin, in 20 patients with AQP4 + NMOSD and 17 healthy controls (HCs). Disease controls included 18 patients with MOGAD. Serum and cerebrospinal fluid interleukin-6 levels were also measured. Clinical severity was evaluated with Kurtzke's Expanded Disability Status Scale (EDSS). Compared with HCs, patients with AQP4 + NMOSD showed higher BMP-9 (median; 127 vs. 80.7 pg/mL; P = 0.0499) and leptin levels (median; 16,081 vs. 6770 pg/mL; P = 0.0224), but not those with MOGAD. Better improvement in EDSS at 6 months was associated with baseline BMP-9 levels in patients with AQP4 + NMOSD (Spearman's rho = - 0.47; P = 0.037). Serum BMP-9 is upregulated at relapse and may contribute to vascular remodeling in AQP4 + NMOSD. Serum BMP-9 levels could predict clinical recovery 6 months after the attack.

Delayed Appearance of Brain Magnetic Resonance Imaging Abnormalities in a Patient with Glial Fibrillary Acidic Protein Astrocytopathy.

Recent studies have reported that autoantibodies against glial fibrillary acidic protein (GFAP), a major cytoskeletal protein expressed in astrocytes, can lead to GFAP astrocytopathy, an autoimmune central nervous system inflammatory disease. We herein report the unique case of a 59-year-old Japanese woman with GFAP astrocytopathy who presented with characteristic symptoms, including signs of meningeal irritation, cerebellar ataxia, and bladder/rectal dysfunction, in the absence of specific findings on initial brain magnetic resonance imaging (MRI). The patient exhibited new abnormal changes mainly in the brainstem on follow-up MRI, illustrating the need to recognize that MRI abnormalities may appear later in GFAP astrocytopathy.

Higher longitudinal brain white matter atrophy rate in aquaporin-4 IgG-positive NMOSD compared with healthy controls.

We aimed to compare longitudinal brain atrophy in patients with neuromyelitis optica spectrum disorder (NMOSD) with healthy controls (HCs). The atrophy rate in patients with anti-aquaporin-4 antibody-positive NMOSD (AQP4 + NMOSD) was compared with age-sex-matched HCs recruited from the Japanese Alzheimer's Disease Neuroimaging Initiative study and another study performed at Chiba University. Twenty-nine patients with AQP4 + NMOSD and 29 HCs were enrolled in the study. The time between magnetic resonance imaging (MRI) scans was longer in the AQP4 + NMOSD group compared with the HCs (median; 3.2 vs. 2.9 years, P = 0.009). The annualized normalized white matter volume (NWV) atrophy rate was higher in the AQP4 + NMOSD group compared with the HCs (median; 0.37 vs. - 0.14, P = 0.018). The maximum spinal cord lesion length negatively correlated with NWV at baseline MRI in patients with AQP4 + NMOSD (Spearman's rho = - 0.41, P = 0.027). The annualized NWV atrophy rate negatively correlated with the time between initiation of persistent prednisolone usage and baseline MRI in patients with AQP4 + NMOSD (Spearman's rho = - 0.43, P = 0.019). Patients with AQP4 + NMOSD had a greater annualized NWV atrophy rate than HCs. Suppressing disease activity may prevent brain atrophy in patients with AQP4 + NMOSD.

Roles of lactoferrin on skin wound healing.

Skin wound healing is a complex biological process that requires the regulation of different cell types, including immune cells, keratinocytes, fibroblasts, and endothelial cells. It consists of 5 stages: hemostasis, inflammation, granulation tissue formation, re-epithelialization, and wound remodeling. While inflammation is essential for successful wound healing, prolonged or excess inflammation can result in nonhealing chronic wounds. Lactoferrin, an iron-binding glycoprotein secreted from glandular epithelial cells into body fluids, promotes skin wound healing by enhancing the initial inflammatory phase. Lactoferrin also exhibits anti-inflammatory activity that neutralizes overabundant immune response. Accumulating evidence suggests that lactoferrin directly promotes both the formation of granulation tissue and re-epithelialization. Lactoferrin stimulates the proliferation and migration of fibroblasts and keratinocytes and enhances the synthesis of extracellular matrix components, such as collagen and hyaluronan. In an in vitro model of wound contraction, lactoferrin promoted fibroblast-mediated collagen gel contraction. These observations indicate that lactoferrin supports multiple biological processes involved in wound healing.

Clinical difference after the first optic neuritis between aquaporin-4-IgG-associated and myelin oligodendrocyte glycoprotein-IgG-associated disorders.

OBJECTIVE: To investigate the difference in clinical course after the first optic neuritis (ON) between aquaporin-4 IgG-associated disorder (AQPAD) and myelin oligodendrocyte glycoprotein-IgG-associated disorder (MOGAD) METHODS: In this study, 31 eyes in 24 patients with AQPAD and 26 eyes in 18 patients with MOGAD were included. The clinical course for the first 6 months after the first ON was monitored by a retrospective cohort study. Best-corrected visual acuity (BCVA) was observed before the onset and at nadir, 2 weeks (2 W), 1 month (1 M), 2 months (2 M), 3 months (3 M) and 6 months (6 M). The decimal BCVA was converted to the logarithm of the minimal angle of resolution (logMAR) for statistical analyses. RESULTS: MOGAD eyes showed longer median number of days from ON onset to nadir (6.0 vs. 11.5, P = 0.012) and to treatment (7.0 vs. 11.0, P = 0.020) than AQPAD eyes. The median logMAR was higher in AQPAD eyes than in MOGAD eyes at nadir (2.00 vs. 1.77, P = 0.050), 2 W (1.85 vs. 0.40, P = 0.001), 2 M (0.023 vs. - 0.079, P = 0.032) and 3 M (0.046 vs. - 0.079, P = 0.002). The median time to recovery of BCVA to 0.7 was longer in AQPAD eyes than in MOGAD eyes (44.0 vs. 21.0 days, P = 0.024), but that to BCVA 1.0 was not different between the two disorders (168.0 vs. 40.0 days, respectively, P = 0.056). CONCLUSION: Compared with MOGAD eyes, AQPAD eyes tended to show worse visual outcome even during the first ON episode.

Reduced fucosylation in the distal intestinal epithelium of mice subjected to chronic social defeat stress.

Psychological stress can cause dysfunction of the gastrointestinal tract by regulating its interaction with central nervous system (brain-gut axis). Chronic social defeat stress (CSDS) is widely used to produce a rodent model of stress-induced human mood disorders and depression. We previously showed that CSDS significantly affects the intestinal ecosystem including cecal and fecal microbiota, intestinal gene expression profiles and cecal metabolite profiles. Here, we investigated whether the glycosylation pattern in the intestinal epithelium was affected in C57BL/6 mice exposed to CSDS (hereinafter referred to as CSDS mice). A lectin microarray analysis revealed that CSDS significantly reduced the reactivity of fucose-specific lectins (rAOL, TJA-II, rAAL, rGC2, AOL, AAL, rPAIIL and rRSIIL) with distal intestinal mucosa, but not with mucosa from proximal intestine and colon. Flow cytometric analysis confirmed the reduced TJA-II reactivity with intestinal epithelial cells in CSDS mice. In addition, distal intestine expression levels of the genes encoding fucosyltransferase 1 and 2 (Fut1 and Fut2) were downregulated in CSDS mice. These findings suggest that CSDS alters the fucosylation pattern in the distal intestinal epithelium, which could be used as a sensitive marker for CSDS exposure.

The distinct effects of orally administered Lactobacillus rhamnosus GG and Lactococcus lactis subsp. lactis C59 on gene expression in the murine small intestine.

The molecular mechanisms of strain-specific probiotic effects and the impact of the oral administration of probiotic strains on the host's gene expression are not yet well understood. The aim of this study was to investigate the strain-specific effects of probiotic strain intake on gene expression in the murine small intestine. Two distinct strains of lactic acid bacteria, Lactobacillus rhamnosus GG (GG) and Lactococcus lactis subsp. lactis C59 (C59), were orally administered to BALB/c mice, daily for 2 weeks. The total RNA was isolated from the upper (including the duodenum) and lower (the terminal ileum) small intestine, and gene expression was assessed by microarray analysis. The data revealed (1) oral administration of C59 and GG markedly down-regulated the expression of genes encoding fibrinogen subunits and plasminogen in the upper small intestine; (2) administration of more than 1 × 107 CFU/day of GG changed the gene expression of the host ileum. (3) strain- and dose-related effects on various GO biological processes; and (4) enrichment for B cell-related Gene Ontology terms among up-regulated genes in the terminal ileum of mice administered the 1 × 109 CFU/day of GG. The distinct effects of GG and C59 on gene expression in the intact small intestine provide clues to understand how the health beneficial effects of specific strains of probiotic bacteria are mediated by interactions with intestinal cells.

First Complete Genome Sequence of the Skin-Improving Lactobacillus curvatus Strain FBA2, Isolated from Fermented Vegetables, Determined by PacBio Single-Molecule Real-Time Technology.

The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Protective effect of indole-3-pyruvate against ultraviolet b-induced damage to cultured HaCaT keratinocytes and the skin of hairless mice.

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm2) and maintained with or without test compounds (1-25 mM).In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (10 µmol) and exposed to UVB light (1 J/cm2) twice [corrected]. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1ß (IL-1ß) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1ß and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.