Immediate-type allergic and protease-mediated reactions are involved in scratching behaviour induced by topical application of Dermatophagoides farinae extract in NC/Nga mice.

Atopic dermatitis (AD)-like dermatitis can be induced by repeated topical application of an ointment containing Dermatophagoides farinae body (Dfb) extract in NC/Nga mice. This AD-like murine model also exhibits a biphasic increase in the number of scratching behaviour after topical application of Dfb ointment. In this study, we investigated the possible mechanisms underlying the scratching behaviour in each phase. An increase in the content of mast cell-derived mediators such as histamine and 5-hydroxytryptamine in the lesional skin and increased vascular permeability were observed in the early phase after the Dfb ointment application. Chlorpheniramine (H1 receptor antagonist) and cromoglycate (mast cell stabilizer) reduced the scratching behaviour in the early phase but not that in the later phase. Furthermore, the content of various endogenous pruritogens such as interleukin-31 and thymic stromal lymphopoietin in the lesional skin was increased 1 or 24 hours after the Dfb ointment application. Elevated expression of proteinase-activated receptor-2 (PAR-2) was also observed in the epidermis. Finally, gabexate (serine protease inhibitor) reduced the scratching behaviour in both phases, and anti-PAR2 antibody also showed a tendency to reduce both scratching behaviours. These findings suggest that immediate-type allergic reactions caused by mast cell degranulation and PAR-2 activation by proteases are involved in the scratching behaviour in this AD-like model.

Bactericidal activity and post-antibiotic effect of ozenoxacin against Propionibacterium acnes.

Ozenoxacin, a novel non-fluorinated topical quinolone, is used for the treatment of acne vulgaris in Japan. We investigated bactericidal activity and post-antibiotic effect (PAE) of ozenoxacin against Propionibacterium acnes, a major causative bacterium of acne vulgaris. The minimum inhibitory concentrations (MICs) of ozenoxacin against 3 levofloxacin-susceptible strains (MIC of levofloxacin; ≤4 µg/mL) and 3 levofloxacin-resistant strains (MIC of levofloxacin; ≥8 µg/mL) ranged from 0.03 to 0.06 µg/mL and from 0.25 to 0.5 µg/mL, respectively. These MICs of ozenoxacin were almost the same or lower than nadifloxacin and clindamycin. The minimum bactericidal concentrations (MBCs) of ozenoxacin against the levofloxacin-susceptible and -resistant strains were from 0.06 to 8 µg/mL and from 0.5 to 4 µg/mL, respectively. These MBCs were lower than those of nadifloxacin and clindamycin. In time-kill assay, ozenoxacin at 1/4, 1 and 4 times the respective MIC against both levofloxacin-susceptible and -resistant strains showed a concentration-dependent bactericidal activity. Ozenoxacin at 4 times the MICs against the levofloxacin-susceptible strains showed more potent and more rapid onset of bactericidal activity compared to nadifloxacin and clindamycin at 4 times the respective MICs. The PAEs of ozenoxacin at 4 times the MICs against the levofloxacin-susceptible strains were from 3.3 to 17.1 h, which were almost the same or longer than nadifloxacin and clindamycin. In contrast, the PAEs were hardly induced by any antimicrobial agents against the levofloxacin-resistant strains. The present findings suggest that ozenoxacin has a potent bactericidal activity against both levofloxacin-susceptible and -resistant P. acnes, and a long-lasting PAE against levofloxacin-susceptible P. acnes.

Antimicrobial activities of ozenoxacin against isolates of propionibacteria and staphylococci from Japanese patients with acne vulgaris.

Ozenoxacin, a novel non-fluorinated topical quinolone, was assessed for in vitro antimicrobial activity against clinical isolates of propionibacteria and staphylococci according to the broth microdilution method recommended by the Clinical and Laboratory Standards Institute. The isolates used in this study were collected from Japanese patients with acne vulgaris during a period from 2012 to 2013. The MIC90s of ozenoxacin against Propionibacterium acnes (n=266), Propionibacterium granulosum (n=10), Staphylococcus aureus (n=23), Staphylococcus epidermidis (n=229) and other coagulase-negative staphylococci (n=82) were ≤0.06, ≤0.06, ≤0.06, 0.125 and ≤0.06 µg ml-1, respectively. The antimicrobial activity of ozenoxacin against the clinical isolates of propionibacteria and staphylococci was greater than that of five reference antimicrobial agents which have been used for the treatment of acne vulgaris. The MICs of ozenoxacin were correlated with those of nadifloxacin in P. acnes and S. epidermidis isolates. However, the MICs of ozenoxacin were 0.25-0.5 µg ml-1 and 0.5-8 µg ml-1 against nadifloxacin-resistant P. acnes (MIC: ≥8 µg ml-1; n=8) and S. epidermidis (MIC: ≥64 µg ml-1; n=10), respectively. These results indicated the potent antimicrobial activity against P. acnes and S. epidermidis isolates resistant to nadifloxacin. Topical ozenoxacin could represent an alternative therapeutic drug for acne vulgaris based on its potent antimicrobial activity against the isolates of propionibacteria and staphylococci from acne patients.

In vitro antimicrobial activity of ozenoxacin against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus and Streptococcus pyogenes isolated from clinical cutaneous specimens in Japan.

Ozenoxacin, a novel non-fluorinated topical quinolone, was assessed for in vitro antimicrobial activity against each 50 isolates of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and Streptococcus pyogenes according to the broth microdilution method recommended by the Clinical and Laboratory Standards Institute. The isolates used in this study were recovered from cutaneous specimens of Japanese adult and pediatric patients who visited hospitals in 2014. The MIC90s of ozenoxacin against MSSA, MRSA and S. pyogenes isolates from adult patients were ≤0.06, 4 and ≤0.06 µg/mL, respectively. The MIC90s of ozenoxacin against MSSA and S. pyogenes isolates from pediatric patients were equal to those against the adult isolates. On the other hand, the MIC90s of ozenoxacin against the pediatric MRSA isolates was 0.12 µg/mL, and was 32 times lower than that against the adult isolates. The antimicrobial activity of ozenoxacin against MSSA, MRSA and S. pyogenes was equal to or greater than those of 7 reference antimicrobial agents had been used for the treatment of skin infections. The MICs of ozenoxacin was highly correlated with those of nadifloxacin and levofloxacin in the 50 MRSA isolates (r(2) = 0.906 and 0.833, respectively). However, ozenoxacin kept the potent antimicrobial activity with the MIC ranging from 1 to 4 µg/mL even against MRSA low susceptible (MIC: >64 µg/mL) to nadifloxacin or levofloxacin. Ozenoxacin could represent the first-in-class non-fluorinated quinolone for the topical treatment of various superficial skin infections caused by MSSA, MRSA and S. pyogenes.

Biphasic increase in scratching behaviour induced by topical application of Dermatophagoides farinae extract in NC/Nga mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied by severe itching and eczematous lesion. In this study, we applied an ointment containing Dermatophagoides farinae body (Dfb) extract repeatedly on the dorsal skin of NC/Nga mice with barrier disruption to investigate the characteristics of this murine model of human AD. Following repeated topical application of Dfb ointment twice weekly for 2 weeks, the dermatitis score increased gradually, accompanied by an elevation of total immunoglobulin E level in plasma. Topical application of Dfb ointment also caused epidermal hyperplasia and accumulation of inflammatory cells in the lesional skin and increased expression of T-helper (Th) 1/Th2/Th17 cytokines in axillary lymph node cells. Furthermore, increased sprouting of intraepidermal nerve fibres was observed with an increase in the content of nerve growth factor and decrease in that of semaphorin 3A in the lesional skin. These findings suggest that the characteristics in this model were similar to those observed in patients with AD. Interestingly, it was observed for the first time that scratching behaviour increased in a biphasic fashion by topical application of Dfb ointment in addition to an increase in spontaneous scratching behaviour in this model. It is also suggested that further clarifying the underlying mechanisms of scratching behaviour in this model leads not only to elucidating the pathogenesis of AD but also to discovering novel therapeutic drugs for AD.

Analysis of l-DOPA-derived melanin and a novel degradation product formed under alkaline conditions.

When the therapeutic drug l-DOPA, which is used to treat Parkinson's disease, is combined with magnesium oxide (MgO), a formulation change produces a dark substance. Infrared spectroscopy reveals that this substance is melanin. After allowing the l-DOPA and MgO mixture to stand, the l-DOPA content decreases significantly, and a new degradation product (the final degradation product of l-DOPA, FDP-D) is generated. Formation of this product requires a solution with a pH of >10, and the presence of MgO is not necessary. FDP-D is not produced by tyrosinase decomposition of l-DOPA and is therefore not a melanin-related compound. Pure FDP-D is isolated by adjusting the l-DOPA solution to pH 10 with ammonium hydroxide, allowing it to stand for 3 days at room temperature, adding trifluoroacetic acid (TFA), filtering the precipitate, and separating the supernatant with high-performance liquid chromatography (HPLC). Mass spectrometry indicates that the isolated FDP-D has a molecular formula of C9H9NO7. On the basis of NMR analysis ((1)H NMR, (13)C NMR, DEPT, H-H COSY, HMQC, and HMBC), FDP-D appears to be a substance with the novel structure 7a-hydroxy-5-oxo-1,2,3,5,7,7a-hexahydropyrano [3,4-b]pyrrole-2,7-dicarboxylic acid.

In vitro antimicrobial activity of benzoyl peroxide against Propionibacterium acnes assessed by a novel susceptibility testing method.

Benzoyl peroxide (BPO), a therapeutic agent for acne vulgaris, was assessed for in vitro antimicrobial activity against Propionibacterium acnes using a novel broth microdilution testing that improved BPO solubility. We searched for a suitable culture medium to measure the minimum inhibitory concentration (MIC) of BPO against P. acnes and finally found the Gifu anaerobic medium (GAM) broth supplemented with 0.1(v/v)% glycerol and 2(v/v)% Tween 80, in which BPO dissolved up to 1250 µg/mL and P. acnes grew well. The MICs and minimum bactericidal concentrations (MBCs) of BPO against 44 clinical isolates of P. acnes collected from Japanese patients with acne vulgaris were determined by our testing method using the supplemented GAM broth. The MICs of BPO were 128 or 256 µg/mL against all isolates of P. acnes regardless of susceptibility to nadifloxacin or clindamycin. The MBCs of BPO were also 128 or 256 µg/mL against the same isolates. Moreover, BPO at the MIC showed a rapid bactericidal activity against P. acnes ATCC11827 in time-kill assay. In conclusion, we could develop a novel assay for the MIC and MBC determinations of BPO against P. acnes, which is reliable and reproducible as a broth microdilution testing and the present results suggest that BPO has a potent bactericidal activity against P. acnes.

Anti-inflammatory activity of lanoconazole, a topical antifungal agent.

Topical antifungal agents which have anti-inflammatory effects have the potential to provide additional clinical benefits. Therefore, an anti-inflammatory activity of lanoconazole (LCZ), a topical antifungal agent, was investigated against in vitro and in vivo models of inflammation. The release of interleukin-8 (IL-8) from human epidermal keratinocytes stimulated by the addition of 100 µg ml(-1) ß-glucan of Saccharomyces cerevisiae was significantly inhibited by LCZ at the concentration of 10(-5) mol l(-1). The release of interferon-γ and IL-2 from human peripheral blood mononuclear cells stimulated by the addition of 30 and 100 µg ml(-1) phytohemagglutinin was significantly inhibited by LCZ at the concentrations of 10(-7) and 10(-6) mol l(-1), respectively. The increase in the ear thickness induced by topical application of 0.01% 12-O-tetradecanoyl phorbol-13-acetate and 1% 2,4,6-trinitrochlorobenzene (TNCB) after sensitisation with 3% TNCB were established as the mouse models of irritant and contact dermatitis, respectively. Application of 1% and 3% LCZ showed a significant anti-inflammatory activity against both the irritant and contact dermatitis models. These findings suggest that LCZ possesses an anti-inflammatory activity, which may be partially helpful in the treatment of dermatomycoses.

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances.

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8(+) cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D(b) class I MHC-restricted antigens and CD1d-restricted substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4(-)CD8(-) and CD8(+) NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4(-)CD8(-), but not CD8(+) NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.

Surgical therapy in Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) is usually considered to be manageable by levothyroxine (L-T4) administration, which can reduce the thyroid volume and supplement the lack of hormone. However, we sometimes encounter a huge goiter that has not shrunk in response to L-T4 therapy. These goiters continue to produce symptoms of compression and an unsightly appearance. Here we discuss the surgical indication and procedure for HT. Thirteen patients with clinically diagnosed Hashimoto's thyroiditis involving a huge diffuse goiter that produced pressure symptoms or nodular lesions were treated with surgery. The gender, age distribution, total dose and period of L-T4 administration prior to the operation, and clinical symptoms caused by the large goiter were evaluated in each case. The titer of antibodies was extremely elevated in 8 HT patients with a diffusely enlarged goiter. The total period of L-T4 medication ranged from 6 to 25 years. A subtotal thyroidectomy in which a small amount of thyroid was left in the posterior area of the bilateral lobes was performed in the 8 cases of diffusely enlarged goiter. Pressure symptoms and the unsightly appearance caused by the goiter were relieved by the surgery in all cases. No surgical complications developed. In conclusion, the surgery is an effective therapy for HT patients who have persistent compression symptoms and/or an unsightly neck appearance due to a large goiter despite long-term L-T4 treatment.

Evaluation of an alternative, subclavicular approach to thyroidectomy.

BACKGROUND: After presenting with the common finding of a nodular goiter, many patients refuse surgery because of the potential for a conspicuous anterior neck scar. Despite the development of endoscopic approaches to neck dissections, it can be difficult to remove a large thyroid tumor using such approaches. MATERIAL/METHODS: We attempted thyroid lobectomies via a subclavicular approach in three cases - three women whose ages ranged from 36-45 years old. Each patient was diagnosed with a cytologically-benign nodular goiter with a diameter greater than 60 mm. Subclavicular incisions were made with the length being determined by the size of the tumor and with the thyroid being approached laterally for resection. RESULTS: Despite a maximum tumor diameter of greater than 70 mm, our operative technique was comparable to conventional methods with respect to surgical exposure, safety and operation time. Cosmetically, each patient was satisfied with the post-operative results. CONCLUSIONS: We believe that the subclavicular approach to thyroidectomy is a safe and cosmetically-superior alternative to conventional surgery, even in cases that are too large to approach endoscopically.