A rapidly progressive multiple system atrophy-cerebellar variant model presenting marked glial reactions with inflammation and spreading of α-synuclein oligomers and phosphorylated α-synuclein aggregates.

Multiple system atrophy (MSA) is a severe α-synucleinopathy facilitated by glial reactions; the cerebellar variant (MSA-C) preferentially involves olivopontocerebellar fibres with conspicuous demyelination. A lack of aggressive models that preferentially involve olivopontocerebellar tracts in adulthood has hindered our understanding of the mechanisms of demyelination and neuroaxonal loss, and thus the development of effective treatments for MSA. We therefore aimed to develop a rapidly progressive mouse model that recaptures MSA-C pathology. We crossed Plp1-tTA and tetO-SNCA*A53T mice to generate Plp1-tTA::tetO-SNCA*A53T bi-transgenic mice, in which human A53T α-synuclein-a mutant protein with enhanced aggregability-was specifically produced in the oligodendrocytes of adult mice using Tet-Off regulation. These bi-transgenic mice expressed mutant α-synuclein from 8 weeks of age, when doxycycline was removed from the diet. All bi-transgenic mice presented rapidly progressive motor deterioration, with wide-based ataxic gait around 22 weeks of age and death around 30 weeks of age. They also had prominent demyelination in the brainstem/cerebellum. Double immunostaining demonstrated that myelin basic protein was markedly decreased in areas in which SM132, an axonal marker, was relatively preserved. Demyelinating lesions exhibited marked ionised calcium-binding adaptor molecule 1-, arginase-1-, and toll-like receptor 2-positive microglial reactivity and glial fibrillary acidic protein-positive astrocytic reactivity. Microarray analysis revealed a strong inflammatory response and cytokine/chemokine production in bi-transgenic mice. Neuronal nuclei-positive neuronal loss and patchy microtubule-associated protein 2-positive dendritic loss became prominent at 30 weeks of age. However, a perceived decrease in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta in bi-transgenic mice compared with wild-type mice was not significant, even at 30 weeks of age. Wild-type, Plp1-tTA, and tetO-SNCA*A53T mice developed neither motor deficits nor demyelination. In bi-transgenic mice, double immunostaining revealed human α-synuclein accumulation in neurite outgrowth inhibitor A (Nogo-A)-positive oligodendrocytes beginning at 9 weeks of age; its expression was further increased at 10 to 12 weeks, and these increased levels were maintained at 12, 24, and 30 weeks. In an α-synuclein-proximity ligation assay, α-synuclein oligomers first appeared in brainstem oligodendrocytes as early as 9 weeks of age; they then spread to astrocytes, neuropil, and neurons at 12 and 16 weeks of age. α-Synuclein oligomers in the brainstem neuropil were most abundant at 16 weeks of age and decreased thereafter; however, those in Purkinje cells successively increased until 30 weeks of age. Double immunostaining revealed the presence of phosphorylated α-synuclein in Nogo-A-positive oligodendrocytes in the brainstem/cerebellum as early as 9 weeks of age. In quantitative assessments, phosphorylated α-synuclein gradually and successively accumulated at 12, 24, and 30 weeks in bi-transgenic mice. By contrast, no phosphorylated α-synuclein was detected in wild-type, tetO-SNCA*A53T, or Plp1-tTA mice at any age examined. Pronounced demyelination and tubulin polymerisation, promoting protein-positive oligodendrocytic loss, was closely associated with phosphorylated α-synuclein aggregates at 24 and 30 weeks of age. Early inhibition of mutant α-synuclein expression by doxycycline diet at 23 weeks led to fully recovered demyelination; inhibition at 27 weeks led to persistent demyelination with glial reactions, despite resolving phosphorylated α-synuclein aggregates. In conclusion, our bi-transgenic mice exhibited progressively increasing demyelination and neuroaxonal loss in the brainstem/cerebellum, with rapidly progressive motor deterioration in adulthood. These mice showed marked microglial and astrocytic reactions with inflammation that was closely associated with phosphorylated α-synuclein aggregates. These features closely mimic human MSA-C pathology. Notably, our model is the first to suggest that α-synuclein oligomers may spread from oligodendrocytes to neurons in transgenic mice with human α-synuclein expression in oligodendrocytes. This model of MSA is therefore particularly useful for elucidating the in vivo mechanisms of α-synuclein spreading from glia to neurons, and for developing therapies that target glial reactions and/or α-synuclein oligomer spreading and aggregate formation in MSA.

Role of ERK Pathway in the Pathogenesis of Atopic Dermatitis and Its Potential as a Therapeutic Target.

Atopic dermatitis (AD) is an eczematous skin disorder characterized by type 2 inflammation, barrier disruption, and intense itch. In addition to type 2 cytokines, many other cytokines, such as interferon gamma (IFN-γ), interleukin 17 (IL-17), and interleukin 22 (IL-22), play roles in the pathogenesis of AD. It has been reported that the extracellular signal-regulated kinase (ERK) is downstream of such cytokines. However, the involvement of the ERK pathway in the pathogenesis of AD has not yet been investigated. We examined the expression of p-ERK in mouse and human AD skin. We also investigated the effects of the topical application of an ERK inhibitor on the dermatitis score, transepidermal water loss (TEWL), histological change, and expression of filaggrin, using an AD-like NC/Nga murine model. The effects of an ERK inhibitor on filaggrin expression in normal human epidermal keratinocytes (NHEKs) and on chemokine production from bone marrow-derived dendritic cells (BMDCs) were also evaluated. p-ERK was highly expressed in mouse and human AD skin. Topical application of an ERK inhibitor alleviated the clinical symptoms, histological changes, TEWL, and decrease in expression of filaggrin in the AD-like NC/Nga murine model. The ERK inhibitor also restored the IL-4 induced reduction in the expression of filaggrin in NHEK, and inhibited chemokine production from BMDC induced by IL-4. These results indicate that the ERK pathway is involved in the pathogenesis of AD, and suggest that the ERK pathway has potential as a therapeutic target for AD in the future.

Human cathelicidin antimicrobial peptide LL-37 promotes lymphangiogenesis in lymphatic endothelial cells through the ERK and Akt signaling pathways.

LL-37, the only member of the cathelicidin family of cationic antimicrobial peptides in humans has been shown to exhibit a wide variety of biological actions in addition to its antimicrobial activity. However, the lymphangiogenic effect of LL-37 has not been elucidated yet. In this study, we examined the effects of LL-37 on lymphangiogenesis and evaluated the underlying molecular mechanisms. LL-37 treatment significantly increased the migration and tube-like formation of human dermal lymphatic microvascular endothelial cells (HDLECs) and promoted the expression of lymphangiogenic factor in HDLECs. Treatment with LL-37 increased phosphorylation of ERK and Akt proteins in HDLECs, and pretreatment with ERK and Akt inhibitors significantly blocked the LL-37-induced HDLEC migration and tube-like formation. Furthermore, to investigate the involvement of formyl peptide receptor-like 1 (FPRL1) signaling in LL-37-induced lymphangiogenesis, HDLECs were treated with an FPRL1 antagonist. Pretreatment with the FPRL1 antagonist inhibited LL-37-induced phosphorylation of ERK and Akt proteins and attenuated LL-37-induced HDLEC migration and tube-like formation. These data indicated that LL-37 induces lymphangiogenesis in lymphatic endothelial cells via FPRL1, and the activation of the ERK and Akt-dependent signaling pathways.

Topical application of endothelin receptor a antagonist attenuates imiquimod-induced psoriasiform skin inflammation.

Endothelin-1 (ET-1) is well known as the most potent vasoconstrictor, and can evoke histamine-independent pruritus. Recently, its involvement in cutaneous inflammation has begun to draw attention. The upregulation of ET-1 expression in the epidermis of human psoriasis patients has been reported. It was also demonstrated that ET-1 can stimulate dendritic cells to induce Th17/1 immune responses. However, the role of the interaction between ET-1 and ET-1 receptors in the pathogenesis of psoriasis remains elusive. Here, we investigated the effects of ET-1 receptor antagonist on imiquimod (IMQ) -induced psoriasiform dermatitis in mouse. Psoriasis-related cytokines such as IL-17A and TNF-α induced ET-1 expression in human keratinocytes. Topical application of selective endothelin A receptor (ETAR) antagonist ambrisentan significantly attenuated the development of IMQ-induced psoriasiform dermatitis and also significantly inhibited the histological inflammation and cytokine expression (TNF-α, IL-12p40, IL-12 p19, and IL-17) in the lesional skin of the mouse model. Furthermore, topical application of ambrisentan suppressed phenotypic and functional activation of dendritic cells in lymph nodes. Our findings indicate that the ET-1 and ETAR axis plays an important role in the pathogenesis of psoriasis and is a potential therapeutic target for treating psoriasis.

Pathogenic mechanisms of cariogenic Propionibacterium acidifaciens.

OBJECTIVE: Dental caries is one of the most common infectious diseases in humans. Older adults retain more teeth than did earlier generations and thus are at high risk of root caries. The root surface is covered by cementum, which facilitates the spread of caries lesions into dentinal tissues. Propionibacterium acidifaciens has been detected in dentinal caries lesions; however, the pathogenetic mechanisms are not known. The purpose of this study was to investigate the pathogenic mechanisms of cariogenic P. acidifaciens. METHODS: Saliva-induced aggregation of P. acidifaciens cells and adherence of the organism to saliva-coated hydroxyapatite were examined. Coaggregation of P. acidifaciens with other bacterial cells and binding of the organism to collagen were examined. Effect of Streptococcus mutans on the biofilm formation by P. acidifaciens was also examined. In addition, the effects of acids on the growth of P. acidifaciens were evaluated. RESULTS: P. acidifaciens exhibited strong binding to collagen but weak or moderate interaction with salivary proteins. P. acidifaciens showed weak coaggregation with streptococcal strains and Fusobacerium nucleatum. Biofilm formation by P. acidifaciens was inhibited by S. mutans. Moreover, P. acidifaciens tolerated to self-produced acids up to threshold concentrations. CONCLUSIONS: The results suggest that P. acidifaciens can bind to and survive inside dentinal tissue, and its acid production at low pH condition is involved in the development of dentinal caries.

Impact of Type and Duration of Application of Commercially Available Oral Moisturizers on Their Antifungal Effects.

PURPOSE: To examine the impact of oral moisturizer type and application time on antifungal effects. MATERIALS AND METHODS: Seventeen oral moisturizers (7 liquids, 10 gels) and amphotericin B (AMPH-B) were tested. Antifungal effects were evaluated with newly opened moisturizer samples (0 hour) and with samples incubated for 8 hours to simulate contact during sleep. Candida albicans samples (108 cells/ml) were placed into cylindrical holes in 50% trypticase soy agar plates. Antifungal effects were evaluated based on growth-inhibitory zones after 24 hours. Equal quantities of moisturizers showing growth-inhibitory zones were mixed as additional samples. The effects of moisturizer type and application time on growth-inhibitory zones were evaluated with ANOVA. Growth-inhibitory zone sizes were compared with multiple comparisons. RESULTS: Growth-inhibitory zones were found with two liquids, one gel, moisturizer mixtures, and AMPH-B. Significant differences in antifungal effects were found among different moisturizer types and between the 0- and 8-hour groups. The growth-inhibitory zones of the 8-hour group were significantly smaller than those of the 0-hour group. In both the 0- and 8-hour groups, the growth-inhibitory zones of the liquid-gel mixtures were significantly larger than those of other moisturizer types, and were the same size as those of AMPH-B at two concentrations (1.25 and 2.5 µg/ml). Growth-inhibitory zones of individual moisturizers and liquid-liquid mixtures were the same size as those of lower AMPH-B concentrations (0.16, 0.31, and 0.63 µg/ml). CONCLUSION: Our findings suggest that mixing liquid and gel moisturizers improves their antifungal efficiency.

Usefulness of Intratracheal Instillation Studies for Estimating Nanoparticle-Induced Pulmonary Toxicity.

Inhalation studies are the gold standard for the estimation of the harmful effects of respirable chemical substances, while there is limited evidence of the harmful effects of chemical substances by intratracheal instillation. We reviewed the effectiveness of intratracheal instillation studies for estimating the hazards of nanoparticles, mainly using papers in which both inhalation and intratracheal instillation studies were performed using the same nanoparticles. Compared to inhalation studies, there is a tendency in intratracheal instillation studies that pulmonary inflammation lasted longer in the lungs. A difference in pulmonary inflammation between high and low toxicity nanoparticles was observed in the intratracheal instillation studies, as in the inhalation studies. Among the endpoints of pulmonary toxicity, the kinetics of neutrophil counts, percentage of neutrophils, and chemokines for neutrophils and macrophages, heme oxygenase-1 (HO-1) in bronchoalveolar lavage fluid (BALF), reflected pulmonary inflammation, suggesting that these markers may be considered the predictive markers of pulmonary toxicity in both types of study. When comparing pulmonary inflammation between intratracheal instillation and inhalation studies under the same initial lung burden, there is a tendency that the inflammatory response following the intratracheal instillation of nanoparticles is greater than or equal to that following the inhalation of nanoparticles. If the difference in clearance in both studies is not large, the estimations of pulmonary toxicity are close. We suggest that intratracheal instillation studies can be useful for ranking the hazard of nanoparticles through pulmonary inflammation.

Impact of Types of Moisturizer and Humidity on the Residual Weight and Viscosity of Liquid and Gel Oral Moisturizers.

PURPOSE: Oral moisturizers need to be selected based on their material properties. The purpose of this study was to investigate the effects of moisturizer type and humidity on the residual weight and viscosity of oral moisturizers. MATERIALS AND METHODS: The weight and viscosity of 17 oral moisturizers (7 liquid and 10 gel) at baseline and after 8 hours were measured using an incubator maintained at 37°C at either 85% or 40% relative humidity (RH). The rate of change in weight (RCW) and the rate of change in viscosity (RCV) were calculated. Data were analyzed with two-way analysis of variance (ANOVA) and Scheffe's test to evaluate the effect of the type of moisturizer (liquid or gel) and humidity (85% or 40% RH) on RCW and RCV. Pearson's correlation coefficient was used to evaluate the relationship between RCW and RCV. RESULTS: Two-way ANOVA results indicated that the type of moisturizer and RH had a significant effect on RCW and RCV (p < 0.05); however, the interaction between them was not significant. The results of multiple comparisons showed that gel moisturizers had a significantly lower RCW and higher RCV than liquid moisturizers (p < 0.05). The RCW and RCV at 40% RH were significantly higher than those at 85% RH (p < 0.05). There was no correlation between RCW and RCV in the liquid moisturizer group, but a significant negative correlation was found in the gel moisturizer group (pp = 0.01). CONCLUSION: Because viscosity of gel moisturizers increases as weight decreases, selecting gel moisturizers with a minimal change in weight and viscosity would be preferable in the case of a long-time application and severe dry mouth.

ß-cell induction in vivo in severely diabetic male mice by changing the circulating levels and pattern of the ratios of estradiol to androgens.

Previously we have generated transgenic (Tg) mice developing severe diabetes early in life with a profound depletion of ß-cells with ß-cell-directed expression of inducible cAMP early repressor-Iγ. Only male mice continue to demonstrate hyperglycemia throughout life. To investigate this sexual dimorphism, we treated severely diabetic male Tg mice with orchiectomy (ORX) or 17ß-estradiol (E2) pellet implantation alone or in combination with ORX and E2-implantation to change the circulating levels and patterns of the ratio of estradiol to androgens. In the Tg-ORX group, the blood-glucose levels decreased to a certain level within several weeks but never reached the female Tg-control level. In contrast, the Tg-ORX+E2 or Tg-E2 group showed a more rapid drop in blood glucose to the basal level with a substantial increase in ß-cells, thus preventing the occurrence of severe diabetes in the male mice. The ß-cells, not only within islet but also in and adjacent to ducts and scattered ß-cell clusters, were strongly induced by 1 week after treatment, and the islet morphology dramatically changed. Enhanced ß-cell induction in the ducts occurred concomitantly with markedly increased levels of pancreatic duodenal homeobox-1 and related transcription factors. The glucose-lowering and ß-cell-increasing effects were independent of the age at which the treatment is started. These data provide evidence that the circulating level of E2 and the ratio of E2 to T greatly affect the blood glucose levels, the ß-cell induction, and the islet morphology in diabetic male Tg mice. This novel mechanism offers great potential for developing strategies to increase the number of ß-cells in vivo.

dpr and sod in Streptococcus mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H2O2.

Large numbers of bacteria coexist in the oral cavity. Streptococcus sanguinis, one of the major bacteria in dental plaque, produces hydrogen peroxide (H(2)O(2)), which interferes with the growth of other bacteria. Streptococcus mutans, a cariogenic bacterium, can coexist with S. sanguinis in dental plaque, but to do so, it needs a means of detoxifying the H(2)O(2) produced by S. sanguinis. In this study, we investigated the association of three oxidative stress factors, Dpr, superoxide dismutase (SOD), and AhpCF, with the resistance of S. sanguinis to H(2)O(2). The knockout of dpr and sod significantly increased susceptibility to H(2)O(2), while the knockout of ahpCF had no apparent effect on susceptibility. In particular, dpr inactivation resulted in hypersensitivity to H(2)O(2). Next, we sought to identify the factor(s) involved in the regulation of these oxidative stress genes and found that PerR negatively regulated dpr expression. The knockout of perR caused increased dpr expression levels, resulting in low-level susceptibility to H(2)O(2) compared with the wild type. Furthermore, we evaluated the roles of perR, dpr, and sod when S. mutans was cocultured with S. sanguinis. Culturing of the dpr or sod mutant with S. sanguinis showed a significant decrease in the S. mutans population ratio compared with the wild type, while the perR mutant increased the ratio. Our results suggest that dpr and sod in S. mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H(2)O(2) in regulating the expression of Dpr.