Real-world Evidence for the Treatment of Rosacea with Sulfur or Metronidazole Preparation in Japanese Patients.

Introduction: There remains to be lacking real-world evidence for the treatment of rosacea with a topical sulfur preparation (TSP) or topical metronidazole preparation (TMP) among Japanese patients. Therefore, in this study, we examined the effects of TSP and TMP on rosacea in Japanese patients in real-world clinical settings. Methods: This retrospective observational analysis reviewed the medical records of 47 Japanese patients who were treated with TSP or TMP for more than 8 weeks in our clinic. Disease severity was evaluated using the Investigator Global Assessment (IGA) and the visual analog scale (VAS) for itching, burning sensation, flushing, and hypersensitivity before and 8 weeks after the initiation of the intervention. Results: In total, 10 erythematotelangiectatic rosacea (ETR) and 12 papulopustular rosacea (PPR) patients treated with TSP and 12 ETR and 13 PPR patients treated with TMP were analyzed. IGA and VAS scores for itching, burning sensation, flushing, and hypersensitivity were noted to significantly improve in the ETR and PPR patient groups treated with TSP and both groups treated with TMP, except for the VAS score for itching in the TSP-treated ETR group. No significant differences were observed in terms of the improvement rates of IGA, VAS scores, or the prevalence of adverse events between the TSP- and TMP-treated groups. Conclusions: As per our findings, TSP and TMP have similarly favorable effects on both ETR and PPR in Japanese patients in real-world settings.

Efficacy of telepractice, an alternative therapy tool during the coronavirus disease 2019 pandemic, for speech disorders related to congenital anomalies.

Since telepractice regulation does not yet exist in Japan, we assessed telepractice efficacy and the level of satisfaction with telepractice versus that with face-to-face practice (FTFP) in speech therapy to establish effective telepractice in Japan. Changes in the number of therapy sessions and therapy levels were compared between telepractice and FTFP sessions conducted during the study period. Additionally, the patients' parents completed a questionnaire survey regarding telepractice. The mean number of sessions was not significantly different between the two types of therapy; the therapy levels, according to stepwise speech therapy, either increased or remained unchanged. The survey showed satisfaction with telepractice among all parents. Telepractice for cleft palate speech was delivered successfully with complete parental satisfaction.

Characteristics of Rosacea and Similar Diseases in Patients Wearing Face Masks.

Introduction: The present study aimed to obtain fundamental data, including climate conditions and Demodex mites, on rosacea and similar diseases in the situation where the wearing of face masks is mandatory due to the coronavirus disease 2019 pandemic. Methods: We enrolled 86 Japanese patients habitually wearing face masks with rosacea and similar diseases. Disease severity was assessed using the Investigator Global Assessment. The presence of Demodex mites was examined microscopically. Treatment involved acaricidal and antibiotic agents. Results: The numbers of male and female patients enrolled were 11 and 75, respectively. Among these patients, 85 (98.8%), 57 (66.3%), and 76 (88.4%) had rosacea, rosacea-like dermatitis (RLD), and demodicosis, respectively. The monthly number of patients with rosacea and demodicosis showed two peaks from May to June and in October, during which monthly mean temperature was approximately 20°C (68°F). Improvement rates in rosacea, RLD, and demodicosis were significantly higher when Demodex mites were no longer detected after treatment. Conclusion: The present results suggest that a season with a mean temperature of approximately 20°C is a risk factor for rosacea and similar diseases in individuals wearing face masks in Japan, and a decrease in Demodex mites is associated with the attenuation of symptoms.

Visualization of mechanical stress-mediated Ca2+ signaling in the gut using intravital imaging.

Mechanosensory systems have been implicated in the maintenance of gut homeostasis, but details on the related mechanisms are scarce. Recently, we generated a conditional Ca2+ biosensor yellow cameleon 3.60 (YC3.60)-expressing transgenic mouse model and established a five-dimensional (5D; x, y, z, time, and Ca2+) intravital imaging system for investigating lymphoid tissues and enteric epithelial cell responses. To validate this gut-sensing system, we visualized responses of enteric nervous system (ENS) cells in Nestin-Cre/YC3.60flox mice with specific YC3.60 expression. The ENS, including the myenteric (Auerbach's) and submucous (Meissner's) plexuses, could be visualized without staining in this mouse line, indicating that the probe produced sufficient fluorescent intensity. Furthermore, the myenteric plexus exhibited Ca2+ signaling during peristalsis without stimulation. Nerve endings on the surface of enteric epithelia also exhibited Ca2+ signaling without stimulation. Mechanical stress induced transient salient Ca2+ flux in the myenteric plexus and in enteric epithelial cells in the Nestin-Cre/YC3.60 and the CAG-Cre/YC3.60 lines, respectively. Furthermore, the potential TRPM7 inhibitors were shown to attenuate mechanical stress-mediated Ca2+ signaling. These data indicate that the present intravital imaging system can be used to visualize mechanosensory Ca2+ signaling in ENS cells and enteric epithelial cells.

Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo.

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer's patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

Lipid peroxides as endogenous oxidants forming 8-oxo-guanosine and lipid-soluble antioxidants as suppressing agents.

The oxidation of guanosine to 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA is closely associated with induction of various diseases, but the endogenous oxidant species involved remains unclear. Hydrogen peroxides (H2O2) have been considered to be the oxidant, while lipid peroxides are another possible oxidant because generated easily in bio-membranes surrounding DNA. The oxidant potency was compared between lipid peroxides and H2O2. Linoleic acid hydroperoxides (LOOH) formed 8-oxo-dG at a higher level than H2O2 in guanosine or double-stranded DNA. In the presence of a physiological concentration of Fe(2+) to produce hydroxyl radicals, LOOH was also a stronger oxidant. In a lipid micelle, LOOH markedly produced 8-oxo-dG at a concentration one-tenth of that of H2O2. Upon adding to rat hepatic mitochondria, phosphatidylcholine hydroperoxides produced 8-oxo-dG abundantly. Employing HepG2 cells after pretreated with glutathione peroxidase inhibitor, LOOH formed 8-oxo-dG more abundantly than H2O2. Then, antioxidants to suppress the 8-oxo-dG formation were examined, when the nuclei of pre-incubated HepG2 with antioxidants were exposed to LOOH. Water-soluble ascorbic acid, trolox, and N-acetyl cysteine showed no or weak antioxidant potency, while lipid-soluble 2,6-dipalmitoyl ascorbic acid, α-tocopherol, and lipid-soluble phytochemicals exhibited stronger potency. The present study shows preferential formation of 8-oxo-dG upon LOOH and the inhibition by lipid-soluble antioxidants.

(-)-Epigallocatechin-3-gallate induces secretion of anorexigenic gut hormones.

The anorexigenic gut hormones, cholecystokinin (CCK), glucagon-like peptide (GLP)-1 and peptide tyrosine-tyrosine (PYY), are released in response to food intake from the intestines. Dietary nutrients have been shown to stimulate these hormones. Some non-nutrients such as polyphenols show anorexigenic effects on humans. In the present study, we examined whether dietary polyphenols can stimulate secretion of these gut hormones. Caco-2 cells expressed mRNA of the gut hormones, CCK, PC1 (prohormone convertase 1), GCG (glucagon) and PYY. CCK, GLP-1 and PYY were secreted from Caco-2 cells after adding sugars, amino acids or fatty acids. Using Caco-2 cells, epigallocatechin-3-gallate (EGCG), chlorogenic acid and ferulic acid induced secretion of anorexigenic gut hormones. Particularly, EGCG induced secretion of all three hormones. In an ex vivo assay using murine intestines, EGCG also released CCK from the duodenum, and GLP-1 from the ileum. These results suggest that EGCG may affect appetite via gut hormones.

The combined effect of clothianidin and environmental stress on the behavioral and reproductive function in male mice.

Neonicotinoids, some of the most widely used pesticides in the world, act as agonists to the nicotinic acetylcholine receptors (nAChRs) of insects, resulting in death from abnormal excitability. Neonicotinoids unexpectedly became a major topic as a compelling cause of honeybee colony collapse disorder, which is damaging crop production that requires pollination worldwide. Mammal nAChRs appear to have a certain affinity for neonicotinoids with lower levels than those of insects; there is thus rising concern about unpredictable adverse effects of neonicotinoids on vertebrates. We hypothesized that the effects of neonicotinoids would be enhanced under a chronic stressed condition, which is known to alter the expression of targets of neonicotinoids, i.e., neuronal nAChRs. We performed immunohistochemical and behavioral analyses in male mice actively administered a neonicotinoid, clothianidin (CTD; 0, 10, 50 and 250 mg/kg/day), for 4 weeks under an unpredictable chronic stress procedure. Vacuolated seminiferous epithelia and a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 were observed in the testes of the CTD+stress mice. In an open field test, although the locomotor activities were not affected, the anxiety-like behaviors of the mice were elevated by both CTD and stress. The present study demonstrates that the behavioral and reproductive effects of CTD become more serious in combination with environmental stress, which may reflect our actual situation of multiple exposure.

Transgenic labeling of higher order neuronal circuits linked to phospholipase C-ß2-expressing taste bud cells in medaka fish.

The sense of taste plays a pivotal role in the food-selecting behaviors of vertebrates. We have shown that the fish ortholog of the phospholipase C gene (plc-ß2) is expressed in a subpopulation of taste bud cells that transmit taste stimuli to the central nervous system to evoke favorable and aversive behaviors. We generated transgenic medaka expressing wheat germ agglutinin (WGA) under the control of a regulatory region of the medaka plc-ß2 gene to analyze the neuronal circuit connected to these sensory cells. Immunohistochemical analysis of the transgenic fish 12 days post fertilization revealed that the WGA protein was transferred to cranial sensory ganglia and several nuclei in the hindbrain. WGA signals were also detected in the secondary gustatory nucleus in the hindbrain of 3-month-old transgenic fish. WGA signals were observed in several diencephalic and telencephalic regions in 9-month-old transgenic fish. The age-dependent increase in the labeled brain regions strongly suggests that labeling occurred at taste bud cells and progressively extended to cranial nerves and neurons in the central nervous system. These data are the first to demonstrate the tracing of higher order gustatory neuronal circuitry that is associated with a specific subpopulation of taste bud cells. These results provide insight into the basic neuronal architecture of gustatory information processing that is common among vertebrates.

Transient receptor potential channel M5 and phospholipaseC-beta2 colocalizing in zebrafish taste receptor cells.

In mammals, transient receptor potential (TRP) channel M5 (TRPM5) is coexpressed with phospholipaseC-beta2 (PLC-beta2) in the taste receptor cells, and both PLC-beta2 and TRPM5 are essential elements in the signal transduction of sweet, bitter and umami stimuli. In this study, we identified the zebrafish homologue of TRPM5 (zfTRPM5) and examined its expression in the gustatory system by in-situ hybridization. Using a transgenic zebrafish line that expressed green fluorescent protein under the control of the PLC-beta2 promoter, we showed that zfTRPM5 is expressed in green fluorescent protein-labeled cells of the taste buds. These results demonstrate that zfTRPM5 and PLC-beta2 colocalize in zebrafish taste receptor cells, suggesting their crucial roles in taste signaling via the fish taste receptors.

Characterization of ligands for fish taste receptors.

Recent progress in the molecular biology of taste reception has revealed that in mammals, the heteromeric receptors T1R1/3 and T1R2/3 respond to amino acids and sweeteners, respectively, whereas T2Rs are receptors for bitter tastants. Similar taste receptors have also been characterized in fish, but their ligands have not been identified yet. In the present study, we conducted a series of experiments to identify the fish taste receptor ligands. Facial nerve recordings in zebrafish (Danio rerio) demonstrated that the fish perceived amino acids and even denatonium, which is a representative of aversive bitter compounds for mammals and Drosophila. Calcium imaging analysis of T1Rs in zebrafish and medaka fish (Oryzias latipes) using an HEK293T heterologous expression system revealed that both T1R1/3 and a series of T1R2/3 responded to amino acids but not to sugars. A triple-labeling, in situ hybridization analysis demonstrated that cells expressing T1R1/3 and T1R2/3s exist in PLCbeta2-expressing taste bud cells of medaka fish. Functional analysis using T2Rs showed that zfT2R5 and mfT2R1 responded to denatonium. Behavior observations confirmed that zebrafish prefer amino acids and avoid denatonium. These results suggest that, although there may be some fish-specific way of discriminating ligands, vertebrates could have a conserved gustatory mechanism by which T1Rs and T2Rs respond to attractive and aversive tastants, respectively.

[Development of the Telephone Interview for Cognitive Status (TICS) in Japanese].

In recent years, population of elder people has increased in Japan, following augmentation of the number of people with dementia in Japan. Then it is important to detect cognitive impairment in early stage for adequate treatment, care and prevention. We studied 135 subjects, 49 patients with Alzheimer's disease (AD) and 86 healthy controls using Telephone Interview for Cognitive Status (TICS), and developing Japanese version of the TICS (TICS-J). The sensitivity and the specificity of the TICS-J to differentiate AD patients from healthy controls was 98.0% and 90.7%, respectively. Pearson's correlation coefficiency between the TICS-J and Mini-Mental State Examination (MMSE) was 0.858 (p < 0.001). On the receiver operating curves, the area under the curve for the TICS-J was 98.7% (95% CI: 97.5%-100%). These results indicate that TICS-J is sensitive and specific instrument for differentiating AD patients from healthy controls.

[Development of the Telephone Interview for Cognitive Status (TICS) in Japanese].

In recent years, population of elder people has increased in Japan, following augmentation of the number of people with dementia in Japan. Then it is important to detect cognitive impairment in early stage for adequate treatment, care and prevention. We studied 135 subjects, 49 patients with Alzheimer's disease (AD) and 86 healthy controls using Telephone Interview for Cognitive Status (TICS), and developing Japanese version of the TICS (TICS-J). The sensitivity and the specificity of the TICS-J to differentiate AD patients from healthy controls was 98.0% and 90.7%, respectively. Pearson's correlation coefficiency between the TICS-J and Mini-Mental State Examination (MMSE) was 0.858 (p < 0.001). On the receiver operating curves, the area under the curve for the TICS-J was 98.7% (95% CI: 97.5%-100%). These results indicate that TICS-J is sensitive and specific instrument for differentiating AD patients from healthy controls.

Transgenic labeling of taste receptor cells in model fish under the control of the 5'-upstream region of medaka phospholipase C-beta 2 gene.

Vertebrate taste receptor cells express signaling molecules such as taste receptors and effectors to convert taste stimuli to inner cellular signals. Phospholipase C-beta2 (PLC-beta2) is an effector enzyme that is necessary to transduce taste signals in the mouse. It was shown that a subset of the plc-beta2 expressing cells also express taste receptor molecules, T1Rs or T2Rs, in mammals and fish. To label plc-beta2 expressing cells in the model fish species, we constructed a transgene by linking the 5'-upstream region of the medaka plc-beta2 gene to a green fluorescent protein (GFP) gene. The resulting transgenic medaka exhibited GFP signals in taste buds of the lips and the pharyngeal region. Detailed observation revealed that the GFP signals were in a subpopulation of taste bud cells, and co-localized with the transcript of endogenous plc-beta2 gene. Zebrafish introduced with the same transgene showed GFP signals in a subpopulation of taste bud cells of the lips and the pharyngeal region as in the case of medaka. This is the first report of successful labeling of taste receptor cells in two model fish species under the control of the plc-beta2 promoter. This promoter will be a useful genetic tool to study the vertebrate taste system in general.

Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes.

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi (yan) and kazura (kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.

Phospholipase C-beta 2 as a mammalian taste signaling marker is expressed in the multiple gustatory tissues of medaka fish, Oryzias latipes.

Phospholipase C-beta 2 (PLC-beta 2) is a key enzyme in mammalian taste signal transduction. To analyze the taste system in fishes at molecular level, we cloned mfplc-beta 2 as a medaka fish homologue of PLC-beta 2. In situ hybridization analysis revealed that mfplc-beta 2 is expressed in the lip and branchial region where chemosensory tissues are distributed. Immunohistochemical detection of nerve fibers near the mfplc-beta 2 positive cells suggests the characteristic of peripheral sensory cells. These results suggest that mfplc-beta 2 is expressed in the gustatory sensory cells of medaka. This may provide a molecular basis for the taste reception at multiple tissues in fish species.