Kumi Orita

Inducible nitric oxide synthase (iNOS) and α-melanocyte-stimulating hormones of iNOS origin play important roles in the allergic reactions of atopic dermatitis in mice.

Abstract:  To elucidate the possible involvement of nitric oxide (NO) derived from inducible NO synthase (iNOS) in the pathogenesis of patients with allergic rhinitis, we used an animal model of atopic dermatitis (AD) induced by epicutaneous sensitization and analysed the differences in ear thickness, the frequency of scratching and plasma levels of ovalbumin‐specific immunoglobulin E (OVA‐IgE), transforming growth factor (TGF)‐β, tumor necrosis factor (TNF)‐α, adrenocorticotropic hormone (ACTH) and α‐melanocyte‐stimulating hormone (α‐MSH) between control and iNOS−/− mice. Eight‐week‐old control and iNOS−/− male C57BL/6j mice were sensitized three times with OVA antigen. Before and after the last skin sensitization, the number of scratching incidents and the thickness of the ear were examined, and the plasma levels of OVA‐IgE, α‐MSH, ACTH, TGF‐β and TNF‐α were analysed by ELISA. Sensitization of mice with OVA resulted in increased plasma levels of OVA‐IgE, α‐MSH, ACTH, TGF‐β and TNF‐α in control, but not in iNOS−/− mice. The administration of l‐nitro‐arginine‐methyl ester (l‐NAME) abolished all the above changes that occurred in the control mice. In addition, iNOS−/− mice given α‐MSH exhibited a change similar to that seen in the control, whereas iNOS−/− mice given ACTH, TGF‐β or TNF‐α did not demonstrate any changes. These results indicate that symptoms of AD such as scratching can be exacerbated by α‐MSH, which is induced by iNOS‐derived NO.

Strong exercise stress exacerbates dermatitis in atopic model mice, NC/Nga mice, while proper exercise reduces it.

Please cite this paper as: Strong exercise stress exacerbates dermatitis in atopic model mice, NC/Nga mice, while proper exercise reduces it. Experimental Dermatology 2010; 19: 1067–1072.

Dynamic aspects of ascorbic acid metabolism in the circulation: analysis by ascorbate oxidase with a prolonged in vivo half-life.

Because AA (L-ascorbic acid) scavenges various types of free radicals to form MDAA (monodehydroascorbic acid) and DAA (dehydroascorbic acid), its regeneration from the oxidized metabolites is critically important for humans and other animals that lack the ability to synthesize this antioxidant. To study the dynamic aspects of AA metabolism in the circulation, a long acting AOase (ascorbate oxidase) derivative was synthesized by covalently linking PEG [poly(ethylene glycol)] to the enzyme. Fairly low concentrations of the modified enzyme (PEG-AOase) rapidly decreased AA levels in isolated fresh plasma and blood samples with a concomitant increase in their levels of MDAA and DAA. In contrast, relatively high doses of PEG-AOase were required to decrease the circulating plasma AA levels of both normal rats and ODS (osteogenic disorder Shionogi) rats that lack the ability to synthesize AA. Administration of 50 units of PEG-AOase/kg of body weight rapidly decreased AA levels in plasma and the kidney without affecting the levels in other tissues, such as the liver, brain, lung, adrenal grand and skeletal muscles. PEG-AOase slightly, but significantly, decreased glutathione (GSH) levels in the liver without affecting those in other tissues. Suppression of hepatic synthesis of GSH by administration of BSO [L-buthionin-(S,R)-sulfoximine] enhanced the PEG-AOase-induced decrease in plasma AA levels. These and other results suggest that the circulating AA is reductively regenerated from MDAA extremely rapidly and that hepatic GSH plays important roles in the regeneration of this antioxidant.

Prevention of scattered light-induced asthenopia and fatigue by a polarized filter

Background: It has been well documented that a long‐time irradiation of the eye by a strong light elicits eyestrain and fatigue. To elucidate the mechanism for the induction of light‐induced fatigue and asthenopia, changes in the mouse were analyzed after white light‐irradiation to the eye.

Inducible nitric oxide synthase plays important roles in allergic reactions of pollinosis in mice sensitized with pollen allergy.

To elucidate the possible involvement of nitric oxide (NO) derived from inducible NO-synthase (iNOS) in the pathogenesis of patients with allergic rhinitis, we analyzed changes in the frequency of sneezing, plasma levels of NO metabolites, α-melanocyte-stimulating hormone (MSH) and immunoglobulin E and tracheal expression of IgA and mast cell tryptase in control and iNOS−/− mice. Eight-week-old control and iNOS−/− male C57BL/6j mice were sensitized with Cry j I antigen. After the last intranasal challenge of antigen, changes in the frequency of sneezing and plasma levels of IgE, α-MSH and NO metabolites and tracheal expression of iNOS, IgA and mast cell tryptase were analyzed by ELISA and immunohistochemistry using specific antibodies. The sensitization of mice with Cry j I antigen increased plasma levels of NO metabolites, α-MSH and IgE and tracheal expression of iNOS, IgA and mast cell tryptase in control not but in iNOS−/− mice. Administration of NG-nitro-L-arginine methyl ester strongly inhibited all these changes occurred in control mice. These results indicate that the symptom of pollinosis including sneezing is enhanced by iNOS derived NO through activation of α-MSH-receptor containing mast cells enriched with tryptase.

α-Melanocyte-Stimulating Hormone Plays an Important Role in the Onset of Pollinosis in a Pollen Allergy Mouse Model

Background: α-Melanocyte-stimulating hormone (α-MSH) is a neuropeptide that controls melanogenesis in pigmentary cells. In addition, its potent immunomodulatory activity has been recently described in cutaneous inflammatory disorders. However the mechanism of such pollen allergies remains to be elucidated. The purpose of this study was to investigate the role of α-MSH in a murine model of pollen allergy. Methods: Eight-week-old male BDF-1 mice were sensitized with Cry j I. After the last intranasal antigen, the number of sneezes was counted for 5 min. In addition, the serum levels of IgE and neuronal hormones were measured by ELISA. The expression of IgA, melanocortin receptor 1 (MC1R) and MC5R in the trachea were also observed by immunohistochemistry. Results: Both the concentration of α-MSH and adrenocorticotropin in plasma increase in pollen allergy model mice. Furthermore, MC5R increased in the trachea; however, MC1R did not increase in the trachea. In addition, the changes in sneezing and IgA expression in the pollen allergy model mice were suppressed by α-MSH antibody treatment, but they remained unchanged after MC1R antagonist (agouti) treatment. Conclusions: These results indicate that sneezing due to pollen allergy is associated with an increased concentration of α-MSH and the expression of MC5R.

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.

The accelerated effect of recombinant human bone morphogenetic protein 2 delivered by β-tricalcium phosphate on tendon-to-bone repair process in rabbit models

BACKGROUND Bone morphogenetic protein 2 (BMP-2) plays an important role in the tendon-to-bone repair process. However, there is no previous literature on acceleration of the tendon-to-bone repair process by BMP-2 delivered by β-tricalcium phosphate (β-TCP). The aim of this study was to investigate the accelerated effect of recombinant human BMP-2 (rhBMP-2) delivered by β-TCP on the tendon-to-bone repair process. METHODS The infraspinatus tendon of elderly female Japanese white rabbits was detached from its insertion site on the humerus. A bone tunnel (4.2 mm) was created at the original insertion site of the tendon, which was repaired using the McLaughlin procedure after filling in β-TCP (porosity 75%) without BMP-2 (control group) or with 10 µg rhBMP-2 (BMP group). The rabbits were sacrificed at the second, fourth, and eighth weeks after surgery for histologic analysis and biomechanical testing. We also evaluated the maturity of the tendon-to-bone junction using the tendon-to-bone maturity score. RESULTS Histologic analysis revealed no significant difference between the groups at 2 and 8 weeks but a more abundant organized fibrocartilage at the tendon-to-bone junction in the BMP group at 4 weeks. The tendon-to-bone maturity score improved sequentially. The interface of the BMP group at 4 weeks had significantly improved biomechanical properties than that of the control group. CONCLUSION The tendon-to-bone repair process was facilitated by the use of rhBMP-2 delivered by β-TCP at 4 weeks.

The origin and distribution of CD68, CD163, and αSMA+ cells in the early phase after meniscal resection in a parabiotic rat model

ABSTRACT We previously reported that circulating peripheral blood-borne cells (PBCs) contribute to early-phase meniscal reparative change. Because macrophages and myofibroblasts are important contributors of tissue regeneration, we examined their origin and distribution in the reparative meniscus. Reparative menisci were evaluated at 1, 2, and 4 weeks post-meniscectomy by immunohistochemistry to locate monocytes and macrophages (stained positive for CD68 and CD163), and myofibroblasts (stained positive for αSMA). Of the total number of cells, 13% were CD68+ at 1 week post-meniscectomy, which decreased to 1% by 4 weeks post-meniscectomy; of these, almost half of CD68+ cells (49.4%: 98.8% as PBCs) were green fluorescent protein (GFP)-positive post-meniscectomy (1, 2, and 4 weeks), indicating that the majority of CD68+ cells were derived from PBCs. Of the total cells, 6% were CD163+ at 1 week post-meniscectomy, which decreased to 1% by week 4. Of the CD163+ cells, the majority were GFP-positive (42.5%: 85.0% as PBCs) after 1 week; however, this decreased significantly over time, which indicates that the majority of CD163+ cells are derived from PBCs during the early phase of meniscal reparative change, but are derived from resident cells at later time points. Of the total cells, 38% were αSMA+ at 1 week post-meniscectomy, which decreased to 3% by 4 weeks. The proportion of GFP-positive αSMA+ cells was 2.8% after 1 week, with no significant change over time, which indicates that the majority of αSMA+ cells originated from resident cells. Here, we describe the origin and distribution of macrophages and myofibroblasts during meniscal reparative change.