Sodium Acetate Enhances Neutrophil Extracellular Trap Formation via Histone Acetylation Pathway in Neutrophil-like HL-60 Cells.

Neutrophil extracellular trap formation has been identified as a new cell death mediator, termed NETosis, which is distinct from apoptosis and necrosis. NETs capture foreign substances, such as bacteria, by releasing DNA into the extracellular environment, and have been associated with inflammatory diseases and altered immune responses. Short-chain fatty acids, such as acetate, are produced by the gut microbiota and reportedly enhance innate immune responses; however, the underlying molecular mechanisms remain unclear. Here, we investigated the effects of sodium acetate, which has the highest SCFA concentration in the blood and gastrointestinal tract, on NETosis by focusing on the mechanisms associated with histone acetylation in neutrophil-like HL-60 cells. Sodium acetate enhanced NETosis, as shown by fluorescence staining with SYTOX green, and the effect was directly proportional to the treatment duration (16-24 h). Moreover, the addition of sodium acetate significantly enhanced the acetylation of Ace-H3, H3K9ace, and H3K14ace. Sodium acetate-induced histone acetylation rapidly decreased upon stimulation with the calcium ionophore A23187, whereas histone citrullination markedly increased. These results demonstrate that sodium acetate induces NETosis via histone acetylation in neutrophil-like HL-60 cells, providing new insights into the therapeutic effects based on the innate immunity-enhancing effect of dietary fiber.

Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I.

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

Neutrophil extracellular trap induction through peptidylarginine deiminase 4 activity is involved in 2,4,6-trinitrobenzenesulfonic acid-induced colitis.

Neutrophil extracellular traps (NETs) are induced in the innate immune response against infectious agents and are also implicated in the pathogenesis of various cancers and autoimmune diseases. Peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginine to citrulline, is also involved in NET formation. In this study, we investigated the pathogenic effect of PAD4 on NETs in inflammatory bowel disease using a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. PAD4-deficient (PAD4KO) mice were generated by CRISPR-Cas9-mediated genomic editing. NETs were triggered in peritoneal neutrophils obtained from wild-type mice by A23187 (a calcium ionophore), but these responses were completely abolished in the PAD4KO mice. Experimental colitis was induced in wild-type and PAD4KO mice via an intrarectal injection of TNBS. TNBS injection resulted in body weight loss, extensive colonic erosion, and ulceration in wildtype mice. However, these responses were significantly attenuated following the administration of Cl-amidine (an inhibitor of pan-PADs) and DNase I (an inhibitor of NET formation), in combination with PAD4KO in mice. TNBS-induced increases in myeloperoxidase activity, inflammatory cytokine expression, and NET formation in the colon were significantly reduced following the administration of Cl-amidine, DNase I injection, and PAD4KO. These findings suggest that NET formation contributes to the pathogenesis of TNBS-induced colitis via PAD4. Thus, PAD4 is a promising target for the treatment of inflammatory bowel disease.

High-Dose Vitamin C Administration Inhibits the Invasion and Proliferation of Melanoma Cells in Mice Ovary.

Several studies have been conducted to explore the anticancer effects of vitamin C (VC). However, the effect of high-dose VC administration on melanoma is still unknown. Therefore, in this study, we investigated the effects of high-dose VC (4 g/kg) on the invasion and proliferation of melanoma cells in various organs of mice. B16 melanoma cells (1 × 106 cells/100 µL) were intravenously injected into the tails of female mice, and VC solution (4 g/kg) was orally administered once a day for 14 d. On the 15th day, samples from the liver, lungs, jejunum, and ovaries were collected and analyzed for invasion and proliferation of melanoma cells. Oral VC administration decreased the number of dihydroxyphenylalanine (DOPA)-positive cells and gp100-positive melanoma cells in the ovaries and suppressed the invasion and proliferation of melanoma. Compared to melanoma-administered mice, macrophage inflammatory protein-2 levels and number of neutrophils were increased in the VC + melanoma-administered mice. Furthermore, the concentrations of VC, iron, and hydrogen peroxide, and the number of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly increased in the ovaries of VC + melanoma-administered mice compared to those of melanoma-administered mice. These results suggest that VC can reduce the invasion and proliferation of melanoma cells in the ovaries, and neutrophils in the ovaries play an important role in achieving this melanoma-suppressive effect.

DNA demethylation increases NETosis.

Neutrophil extracellular traps (NETs) occur during the development of autoimmune diseases, cancer and diabetes. A novel form of cell death that is induced by NETs is called NETosis. Although these diseases are known to have an epigenetic component, epigenetic regulation of NETosis has not previously been explored. In the present study, we investigated the effects of epigenetic change, especially DNA demethylation, on NETosis in neutrophil-like cells differentiated from HL-60 cells, which were incubated for 72 h in the presence of 1.25% DMSO. DMSO-differentiated neutrophil-like cells tended to have increased methylation of genomic DNA. NETosis in the neutrophil-like cells was induced by the treatment with A23187, calcium ionophore, and increased by the addition of the DNMT inhibitor 5-azacytidine (Aza) during differentiation. Interestingly, Aza-stimulated neutrophil-like cell induced NETosis without treatment with A23187. Although reactive oxygen species (ROS), especially superoxide and hypochlorous acid, are important in NETosis induction, treatment with Aza decreased production of ROS, while mitochondria ROS scavenger tended to decrease Aza-induced NETosis. Moreover, the genomic DNA in Aza-stimulated neutrophil-like cell was demethylated, and the expression of peptidylarginine deiminase4 (PAD4) and citrullinated histone H3 (R2+R8+R17) was increased, but myeloperoxidase expression was unaffected. Additionally, PAD4 inhibition tended to decrease Aza-induced NETosis. The DNA demethylation induced by the DNMT inhibitor in neutrophil-like cells enhanced spontaneous NETosis through increasing PAD4 expression and histone citrullination. This study establishes a relationship between NETosis and epigenetics for the first time, and indicates that various diseases implicated to have an epigenetic component might be exacerbated by excessive NETosis also under epigenetic control.

Tranexamic Acid Protects Ovary and Testis Functions and Ameliorates Osteoporosis in Mice.

INTRODUCTION: In a rapidly aging society, the number of people suffering from osteoporosis keeps increasing. However, effective prevention strategies for osteoporosis are not yet currently available. OBJECTIVE: In this study, we examined the ameliorative effects of tranexamic acid on osteoporosis in 24-month-old mice. METHODS: During the study period, mice were orally administered tranexamic acid 3 times per week. RESULTS: Bone mineral density, which is a parameter of osteoporosis, was improved following tranexamic acid administration. In addition, female mice evidenced a stronger phenotypic improvement than male mice. In female mice treated with tranexamic acid, ovary abnormalities were reduced. Furthermore, the levels of transforming growth factor-ß, hyaluronic acid, CD44, reactive oxygen species, and apoptosis, as well as the number of infiltrated neutrophils and macrophages in the ovary were lower than those in the control or solvent-administered mice. In addition, 17ß-estradiol levels in blood increased when compared with the control or solvent-treated mice. In addition, administration of tranexamic acid to 24-month-old male mice decreased the level of apoptosis in the testis. However, the levels of 17ß-estradiol and testosterone in blood increased compared with the control or solvent-administered mice. CONCLUSIONS: The use of tranexamic acid had an ameliorative effect on osteoporosis, possibly by protecting ovaries and testes.

Formation of neutrophil extracellular traps in mitochondrial DNA-deficient cells.

Neutrophil extracellular trap (NET) formation plays an important role in inflammatory diseases. Although it is known that NET formation occurs via NADPH oxidase (NOX)-dependent and NOX-independent pathways, the detailed mechanism remains unknown. Therefore, in this study, we aimed to elucidate the role of mitochondria in NOX-dependent and NOX-independent NET formation. We generated mitochondrial DNA-deficient cells (ρ0 cells) by treating HL-60 cells with dideoxycytidine and differentiated them to neutrophil-like cells. These neutrophil-like ρ0 cells showed markedly reduced NOX-independent NET formation but not NOX-dependent NET formation. However, NET-associated intracellular histone citrullination was not inhibited in ρ0 cells. Furthermore, cells membrane disruption in NOX-dependent NET formation occurred in a Myeloperoxidase (MPO) and mixed lineage kinase domain like pseudokinase (MLKL)-dependent manner; however, cell membrane disruption in NOX-independent NET formation partially occurred in an MLKL-dependent manner. These results highlight the importance of mitochondria in NOX-independent NET formation.

The Role of gp91phox and the Effect of Tranexamic Acid Administration on Hair Color in Mice.

We observed that on long-term breeding, gp91phox-knockout (gp91phox-/-) mice developed white hair. Here, we investigate the origin of this hitherto unexplained phenomenon. Moreover, we investigated the effect of tranexamic acid administration on the hair color in gp91phox-/- mice. We administered tranexamic acid (about 12 mg/kg/day) orally to 9-week-old C57BL/6j (control) and gp91phox-/- mice, thrice a week for 12 months. Compared to control mice, gp91phox-/- mice showed more white hair. However, the concentrations of reactive oxygen species and the levels of interleukin (IL)-1ß and transforming growth factor (TGF)-ß in the skin were lower than those in the control group. Furthermore, increase in white hair was observed in the control mice upon administration of the IL-1ß antagonist. On the other hand, administration of tranexamic acid led to brown colored hair on gp91phox-/- mice. Although tranexamic acid treatment did not alter the expression levels of melanocortin receptor 1 and agouti signaling protein on hair follicles, it increased the expression of mahogunin ring finger protein 1 (MGRN1) and collagen XVII. These results suggested that retention of black hair requires the gp91phox/ROS/IL-1ß/TGF-ß pathway and that elevated levels of MGRN1 and collagen XVII lead to brown hair in gp91phox-/- mice.

17-ß-estradiol enhances neutrophil extracellular trap formation by interaction with estrogen membrane receptor.

Cell death-associated neutrophil extracellular trap formation (NETosis) occurs during various autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, as well as during gestation. Although increasing estrogen concentrations associated with pregnancy might induce NETosis via nuclear estrogen receptor (ERα/ERß), little is known about the mechanisms associated with estrogen-induced NETosis. Here, we investigated the effects of estrogen (17-ß-estradiol; E2) on NETosis, focusing on mechanisms associated with estrogen membrane receptor (GPR30) in neutrophil-like HL-60 cells. Our results show that E2 and the GPR30 agonist G-1 increases level of NETosis and NET formation. Moreover, NETosis-associated intracellular and extracellular histone citrullination and peptidyl arginine deiminase 4 (PAD4) expression were also increased by E2 or G-1 treatment. Furthermore, GPR30 antagonist pre-treatment inhibited increases in NETosis and PAD4 expression mediated by G-1 and partially inhibited these effects mediated by E2. These results demonstrate that E2 treatment induces NETosis via not only ERα/ERß but also GPR30 in neutrophil-like HL-60 cells.

Reduction of oxidative stress a key for enhanced postoperative recovery with fewer complications in esophageal surgery patients: Randomized control trial to investigate therapeutic impact of anesthesia management and usefulness of simple blood test for prediction of high-risk patients.

BACKGROUND: Oxidative stress may be an integral determinant of surgical stress severity. We examined whether the preoperative level of derivatives of reactive oxygen metabolites (d-ROMs), an oxidative stress biomarker based on total hydroperoxides in circulating blood, is predictive of increased risk of delayed recovery and complications after surgery, as well as the effects of anesthesia management on postoperative recovery in light of oxidative stress. METHODS: Patients (American Society of Anesthesiologists physical status I-II) scheduled for a radical esophagectomy (n = 186) were randomly selected to receive inhalational sevoflurane (n = 94) or intravenous propofol (n = 92) anesthesia. Preoperative blood d-ROMs level, as well as pre-and postoperative plasma ferric-reducing ability, were analyzed to assess oxidative stress, with white blood cell (WBC) count, C-reactive protein (CRP) level, incidence of severe postoperative complications, and postoperative recovery process within 30 days after surgery also examined in a double-blind fashion. RESULTS: Postoperative normalization of WBC and CRP was extended in patients with elevated preoperative d-ROMs [WBC versus d-ROMs: correlation coefficient (r) = 0.58 P < .001; CRP versus d-ROMs: r = 0.46 P < .001]. Receiver operating characteristics analysis of d-ROMs in relation to incidence of severe postoperative complications revealed an optimum d-ROMs threshold value of 410 UCarr and that patients with ≥410 UCarr had a greater risk of complications as compared to those with lower values (odds ratio = 4.7). Plasma ferric-reducing ability was decreased by 61 ±â€Š185 mmol·l (P < .001) after surgery, demonstrating development of surgery-related oxidative stress, the magnitude of which was positively correlated with preoperative d-ROMs level (r = 0.16, P = .043). A comparison of the 2 anesthesia management protocols showed that patients who received propofol, an antioxidant anesthetic, had no postoperative decrease in ferric-reducing ability, lower incidence of severe postoperative complications (7 of 92 versus 18 of 94, P = .030, odds ratio = 0.35), and faster uneventful recovery time (WBC normalization days 7.1 ±â€Š5.2 versus 13.6 ±â€Š10.2, P < .001) as compared to those who received sevoflurane. CONCLUSIONS: Elevated preoperative blood d-ROMs predicts greater intraoperative oxidative stress and increased postoperative complications with prolonged recovery, thus is useful for identifying high-risk patients for delayed and complicated surgical recovery. Reduction of oxidative stress is vital for enhanced recovery, with control by antioxidants such as propofol a possible solution.

The Clock Genes Are Involved in The Deterioration of Atopic Dermatitis after Day-and-Night Reversed Physical Stress in NC/Nga Mice.

BACKGROUND: In modern society, irregular lifestyles are a problem. It is well known that Atopic Dermatitis (AD) occurs during physical stress in people with an irregular lifestyle. We evaluated the influence that day-and-night reversal physical stress has on AD. METHODS: Six-week-old specific-pathogen-free and conventional NC/Nga male mice were used. For the day-and-night reversal procedure, the mice ran on a treadmill at a slow speed of 10 m/min for 12 h (between 8:00 and 20:00). Then, between 20:00 and 8:00, we put the mice in a dark place. This treatment was repeated every day for two weeks. The behavioral circadian rhythm of the mice was evaluated with the open field test. Then, the mice were sacrificed and histological examinations of the tissues, the expression of peptide hormones, corticosterone, Immunoglobulin E, histamine, and cytokines was performed using an enzyme-linked immunosorbent assay. RESULTS: In the treadmill-treated conventional NC/Nga mice, AD symptoms were deteriorated compared with the non-treated conventional NC/Nga mice. The levels of Period (Per) 2, Clock, and brain and muscle arnt-like protein 1 (Bmal1) in the skin were increased constantly in the treadmill-treated conventional mice. Furthermore, the expression of Retinoic Acid-related Orphan Receptor (ROR)α, which activates Bmal1, was increased in the treadmill-treated conventional mice compared with the non-treated conventional mice. In addition, when non-treated conventional mice were administrated by the agonist of RORα, AD symptoms were deteriorated similar to treadmill-treated conventional mice. CONCLUSION: In the day-and-night reversal mice, the clock genes were increased constantly, indicating that this is a factor that deteriorated AD.

An Inhibitor of Casein Kinase 1ε/δ (PF670462) Prevents the Deterioration of Dextran Sodium Sulfate-induced Ulcerative Colitis Caused by UVB Eye Irradiation.

Although we previously reported the exacerbation of dextran sodium sulfate (DSS)-induced ulcerative colitis by ultraviolet (UV) B eye irradiation, we do not yet understand the mechanism behind this phenomenon. In this study, we examined the relationship between the deterioration of DSS-induced ulcerative colitis and clock genes. We induced a mouse model of ulcerative colitis by administering DSS for 5 days, and administered UVB eye irradiation on each day of the DSS treatment period. The DSS-induced ulcerative colitis was deteriorated by the UVB eye irradiation. The levels of Clock, brain and muscle arnt-like protein 1 (Bmal1), reverse orientation c-erb A gene (Rev-Erb)α, RAR-related orphan receptor gamma (RORγt), and interleukin (IL)-17 in the colon were increased by UVB eye irradiation in the DSS-treated mice (UVB/DSS-treated mice). Conversely, the nuclear factor, interleukin 3 regulated (NFIL-3) levels in the colon were lower after UVB eye irradiation. The Casein Kinase 1ε/δ inhibitor (PF670462) administration, which is a Clock/Bmal1 inhibitor (PER2 activator), inhibited the deterioration caused by UVB eye irradiation. These results suggest that the UVB eye irradiation-mediated exacerbation of DSS-induced ulcerative colitis depends on IL-17 produced in response to alterations in clock genes.

Gp91phox NADPH oxidase modulates litter size by regulating mucin1 in the uterus of mice.

Active oxygen derived from gp91phox is critical for gestation. However, no reports have evaluated the relationship between reactive oxygen species (ROS) and the number of births in a given pregnancy. In this study, we examined the influence of ROS produced by gp91phox activity on the number of births using C57BL/6j (control) and gp91phox-knockout (gp91phox-/-) mice. The number of births in gp91phox-/- mice was found to be lower than that in control mice. We observed sequential increases in gp91phox, ROS, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), caspase-1, and interleukin-18 (IL-18), followed by increased expression of mucin1 (MUC1), in control mice. However, none of these markers were upregulated in gp91phox-/- mice. In addition, in control mice administered IL-18 or MUC1 inhibitors, the number of births decreased to a number similar to that of gp91phox-/- mice. These results suggest that ROS derived from gp91phox activity altered the inflammatory system and produced IL-18, which subsequently increased the expression of MUC1, thereby modulating fetal development. ABBREVIATIONS: IL-1 ß: interleukin-1ß; IL-18: interleukin-18; NLRP3: nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; IgA: immunoglobulin A; MUC1: mucin1.

The Preventive Effect of Coffee Compounds on Dermatitis and Epidermal Pigmentation after Ultraviolet Irradiation in Mice.

BACKGROUND: Ultraviolet (UV) irradiation is well known to promote inflammation and pigmentation of skin. UVB mainly affects dermatitis and pigmentation. Coffee contains a number of polyphenols, such as caffeic acid (CA) and chlorogenic acid (CGA) but their in vivo bioactivity for photobiology remains unclear. METHODS: C57BL/6j male mice were irradiated with UVB (1.0 kJ/m2/day) for 3 days. Five days after the final session of UVB irradiation, the dorsal skin, ear epidermis, and blood samples were analyzed to investigate the inflammatory factors, melanogenesis factors and related hormones. RESULTS: After the oral administration of CA (100 mg/day) or CGA (100 mg/day) for 8 days, only CA was found to inhibit dermatitis and pigmentation. The pathway by which CA inhibits dermatitis is related to the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK)1/2/cAMP response element binding protein (CREB) pathway. Otherwise, the pathway by which CA inhibits pigmentation is related to the activation of the ß-endorphin-µ-opioid receptor and suppresses the cAMP-microphthalmia-associated transcription factor (MITF) pathway. CONCLUSION: It is suggested that the oral administration of CA prevented dermatitis and pigmentation after UVB irradiation in mice.

Long-Term Ultraviolet A Eye Irradiation Causes Retina Denaturation in Mice.

BACKGROUND: Long-term ultraviolet (UV) A eye irradiation in mice initiates the induction of photoaging. However, the changes in the eye due to long-term exposure to UVA radiation are still unclear. METHODS: Male C57BL/6j (control) and inducible nitric oxide synthase knockout (iNOS-/-) mice were used in this study. The eyes of the mice were locally exposed to UVA radiation for 12 months. RESULTS: The expression of iNOS, matrix metalloproteinase-2 (MMP-2), MMP-9, vascular endothelial growth factor, ß-amyloid, and macrophages in the retina all increased after UVA irradiation. Furthermore, in the iNOS-/- mice, no retinal changes were induced by UVA eye irradiation. CONCLUSIONS: These results indicated that long-term UVA eye irradiation led to iNOS-induced denaturation of the retina; however, further studies are needed to confirm these findings.

Minimal systems analysis of mitochondria-dependent apoptosis induced by cisplatin.

Recently, it was reported that the role of mitochondria-reactive oxygen species (ROS) generating pathway in cisplatin-induced apoptosis is remarkable. Since a variety of molecules are involved in the pathway, a comprehensive approach to delineate the biological interactions of the molecules is required. However, quantitative modeling of the mitochondria-ROS generating pathway based on experiment and systemic analysis using the model have not been attempted so far. Thus, we conducted experiments to measure the concentration changes of critical molecules associated with mitochondrial apoptosis in both human mesothelioma H2052 and their ρ(0) cells lacking mitochondrial DNA (mtDNA). Based on the experiments, a novel mathematical model that can represent the essential dynamics of the mitochondrial apoptotic pathway induced by cisplatin was developed. The kinetic parameter values of the mathematical model were estimated from the experimental data. Then, we have investigated the dynamical properties of this model and predicted the apoptosis levels for various concentrations of cisplatin beyond the range of experiments. From parametric perturbation analysis, we further found that apoptosis will reach its saturation level beyond a certain critical cisplatin concentration.

The Effects of Ultraviolet Eye Irradiation on Dextran Sodium Sulfate-Induced Ulcerative Colitis in Mice.

Ultraviolet (UV) eye irradiation denatures the cells of the intestine. This study examined the action of UVA and UVB on dextran sodium sulfate (DSS)-induced ulcerative colitis. We produced a mouse model of ulcerative colitis by administering DSS for 5 days and irradiated the eye with UVB or UVA for each day of the DSS treatment period. DSS-induced ulcerative colitis was deteriorated by the UVB eye irradiation. Conversely, the symptoms improved with UVA eye irradiation. The levels of adrenocorticotropic hormone (ACTH), corticotropin-releasing hormone (CRH), urocortin 2, interleukin (IL)-18, IL-6 and histamine in the blood increased after the UVB eye irradiation of DSS-treated mice (UVB/DSS-treated mice). In contrast, the ß-endorphin level in the blood of the UVA/DSS-treated mice increased and the levels of urocortin 2, tumor necrosis factor (TNF)-α and histamine decreased. Furthermore, in the colon, the expression of melanocortin-2 receptors (MC2R) increased in the UVB/DSS-treated mice, while the expression of µ-opioid receptors increased in the UVA/DSS-treated mice. When an ACTH inhibitor was administered, UVB eye irradiation caused the deterioration of DSS-treated ulcerative colitis, while the effect of UV eye irradiation disappeared with a µ-opioid receptor antagonist. These results suggested that UV eye irradiation plays an important role in DSS-induced ulcerative colitis.

Neuronal differentiation of human iPS cells induced by baicalin via regulation of bHLH gene expression.

Efficient differentiation is important for regenerative medicine based on pluripotent stem cells, including treatment of neurodegenerative disorders and trauma. Baicalin promotes neuronal differentiation of neural stem/progenitor cells of rats and mice. To evaluate the suitability of baicalin for neuronal differentiation of human iPS cells, we investigated whether it promotes neuronal differentiation in human iPS cells and monitored basic helix-loop-helix (bHLH) gene expression during neuronal differentiation. Baicalin promoted neuronal differentiation and inhibited glial differentiation, suggesting that baicalin can influence the neuronal fate decision in human iPS cells. Notch signaling, which is upstream of bHLH proteins, was not involved in baicalin-induced neuronal differentiation. Baicalin treatment did not down-regulate Hes1 gene expression, but it reduced Hes1 protein levels and up-regulated Ascl1 gene expression. Thus, baicalin promoted neuronal differentiation via modulation of bHLH transcriptional factors. Therefore, baicalin has potential to be used as a small-molecule drug for regenerative treatment of neurodegenerative disorders.

Role of adrenocorticotropic hormone in the modulation of pollinosis induced by pollen antigens.

BACKGROUND: A mild restraint stressor suppressed an increase in the levels of Th2-dependent cytokines and IgE, thereby reducing the symptoms of pollinosis. In the present study, to clarify the mechanism of action of adrenocorticotropic hormone (ACTH) in improving the symptoms of pollinosis, we studied the effects of ACTH on the plasma level of histamine, mast cell number in nasal-associated lymphoid tissue (NALT) and the T cell differentiation in splenocytes. METHODS: The role of ACTH in the development of pollen antigen-induced pollinosis was studied in mice. Allergic symptoms and parameters were measured on day 17 after sensitization. To investigate the effects of ACTH on T cell differentiation, we stimulated splenocytes obtained from control mice with ACTH and CD3/CD28 in vitro, and measured the cytokine production in the culture supernatant. RESULTS: The plasma levels of IL-10, IgE and histamine and mast cell number in NALT were increased in the sensitized animals in association with a concomitant increase in the incidence of sneezing and nasal rubbing. The intraperitoneal administration of ACTH decreased the IL-10, IgE and histamine levels in the plasma and mast cell number in NALT, while increasing the IFN-γ level and suppressing the incidence of nasal rubbing. Furthermore, the production of IFN-γ increased, while the IL-4 level was suppressed after 2 days in culture. CONCLUSIONS: The present findings showed that ACTH directly affects T cell differentiation and promotes Th1-type reactions. The regulation of the Th1/Th2 balance by ACTH may result in a decrease in the pathological features of pollinosis.

UVA irradiation of the eye modulates the contact hypersensitivity of the skin and intestines by affecting mast cells in mice.

BACKGROUND: Ultraviolet A (UVA) irradiation before allergic sensitization induces immunosuppression, but the precise mechanism remained unclear. In this study, we examined the influence of UVA irradiation of the eye on contact hypersensitivity (CHS) and the role of mast cells in CHS. METHODS: We used two types of haptens, fluorescein isothiocyanate (FITC: a Th2 type hapten) and 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone: a Th1 type hapten). A 300 kJ/m(2) dose of UVA irradiation was delivered to the eyes. After UVA irradiation, we sensitized abdominal shaved skin and challenged the ear epidermis and colons of these mice with each hapten. RESULTS: After UVA irradiation, the CHS of the skin and colon were not inhibited in the FITC-sensitized mice. However, in the oxazolone-sensitized mice, only the CHS of the skin was inhibited by UVA irradiation. The inflammation of the colon became more severe after UVA irradiation. In mast cell-deficient (W/Wv) mice sensitized to FITC, the CHS was weaker than that in WT mice. Moreover, the reduction of immunosuppression in ear swelling was seen for one of the two models they used. CONCLUSIONS: These results suggest that the mast cells induced by UVA irradiation of the eye have different roles in the epidermis and colon and have different responses to different haptens.