Clonal deletion of T cell repertoires with specific T cell receptor Vβ chains by two endogenous superantigens in NC/Nga mice

Superantigens (SAgs) are powerful T-cell stimulatory proteins. Because an atopic dermatitis (AD) model NC/Nga mice had two endogenous SAgs, namely minor lymphocyte-stimulating locus-1^a (Mls-1^a) and mouse mammary tumor virus (MMTV)(SHN), SAg-responsive T-cells bearing Vβ5.1, Vβ6, Vβ8.1, Vβ8.2, Vβ8.3, Vβ9, and Vβ11 should be endogenously deleted. Here, we discuss that the endogenous SAgs-expression may be involved in AD-sensitivity in NC/Nga mice.     

Lack of effect of the abnormal fatty acid metabolism in NC/Nga mice on their atopic dermatitis

Although clinical evidence has suggested that dysregulated fatty acid metabolism is associated with atopic disorders, the molecular basis for such a correlation remains to be demonstrated. In the present study, we analyzed the fatty acid composition in peripheral blood cells of NC/Nga mice, a model for atopic dermatitis (AD). We found that arachidonic acid significantly accumulated in mice with the AD manifestation. In addition, the leucotriene B4-releasing ability upon calcium ionophore A23187 stimulation was potentiated in blood cells. An arachidonic acid accumulation was not apparent in the non-atopic BALB/c strain, but was still observed in healthy NC/Nga mice fed under specific pathogen-free conditions. These results indicate that a disturbed fatty acid metabolism in NC/Nga mice was not a trigger factor for their dermatitis development.     

Therapeutic effect of tyndallized Lactobacillus rhamnosus IDCC 3201 on atopic dermatitis mediated by down‐regulation of immunoglobulin E in NC/Nga mice

The therapeutic effect of oral administration of Lactobacillus rhamnosus IDCC 3201 tyndallizate (RHT3201) on atopic dermatitis (AD)‐like skin lesions in NC/Nga mice were investigated. After induction of dermatitis in NC/Nga mice with house‐dust mite extract, each group was fed RHT3201 with 1 × 10⁸, 1 × 10⁹, or 1 × 10¹⁰ cells orally once a day for 8 weeks. Dermatitis scores and frequency of scratching were improved by oral feeding with RHT3201. In contrast to the control group, RHT3201‐fed mice showed significantly down‐regulated mast cell numbers and serum immunoglobulin E (IgE) concentrations had significantly less IL4 in their axillary lymph node cells. The therapeutic effect of RHT3201 was found to be dose‐dependent. These findings indicate that RHT3201 has potential for treating AD.     

Supplemental feeding of phospholipid-enriched alkyl phospholipid from krill relieves spontaneous atopic dermatitis and strengthens skin intercellular lipid barriers in NC/Nga mice

Plasmalogen (Pls) is a glycerophospholipid derived from alkyl phospholipid (Alk) with antioxidant functions in vivo. The present study investigated the effects of ether phospholipids, such as Pls and Alk, on intercellular lipid barriers in the skin of NC/Nga mice, a model of atopic dermatitis (AD). NC/Nga mice fed Alk showed increased plasma levels of Alk and Pls. The AD-related changes in ceramide composition in the skin were abrogated by oral administration of Alk. Moreover, Alk suppressed skin inflammation in AD mice. These results indicate that Alk partially fortifies the stratum corneum lipid barrier and may be an effective treatment for AD.

Abbreviations: Pls: plasmalogen; PlsCho: choline plasmalogen; PlsEtn: ethanolamine plasmalogen; Alk: alkyl phospholipid; TJ: tight junction; FA: fatty acid; AD: atopic dermatitis; SO: soybean oil; FO: fish oil; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TG: triglyceride; PL: phospholipid; RF: retention factor; AlkCho: choline-type alkyl phospholipid; AlkEtn: ethanolamine-type alkyl phospholipid; LC-MS/MS: liquid chromatography-tandem mass spectrometry; FAR1: fatty acyl-coenzyme (Co)A reductase 1     

Orally supplemented Lactobacillus acidophilus strain L‐92 inhibits passive and active cutaneous anaphylaxis as well as 2,4‐dinitroflurobenzene and mite fecal antigen induced atopic dermatitis‐like skin lesions in mice

Oral supplementation of lactic acid bacteria is a potential approach to the prevention and manipulation of allergic diseases such as atopic dermatitis. Our previous report showed that heat‐killed Lactobacillus acidophilus strain L‐92 (L‐92) possessed anti‐allergic properties, although its physiological function in atopic dermatitis has largely remained undefined. To evaluate the anti‐allergic efficacy of L‐92, we used four experimental animal models with the major features of atopic dermatitis and compared the results to those of clinically active drugs. ICR mice were passively sensitized by anti‐dinitrophenyl mouse monoclonal IgE for passive cutaneous anaphylaxis (PCA), and BALB/c mice were actively sensitized by ovalbumin for active cutaneous anaphylaxis (ACA). Allergic reaction was induced by repeated exposure to 2,4‐dinitroflurobenzene (DNFB) and mite (Dermatophagoides farinae) fecal allergen, in BALB/c and NC/Nga mice, respectively. Orally administrated L‐92 significantly inhibited the vascular permeability increase in both PCA and ACA, and the elevation of ovalbumin‐specific IgE titer in ACA. Moreover, repeated applications of DNFB and mite fecal antigen onto the BALB/c and NC/Nga mouse ear, respectively, caused clinical symptoms similar to atopic dermatitis such as ear swelling, scratching behavior and elevation of total serum IgE levels that were also moderately suppressed by L‐92. In addition, L‐92 treated mice exhibited lower levels of mast cells, eosinophil infiltration and Th1/Th2 cytokine expression. Our results, therefore, suggest that oral administration of L‐92 might be useful for alleviating allergic symptoms.     

Effect of dendritic cells treated with CpG ODN on atopic dermatitis of Nc/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease and the pathogenesis of AD is associated with the release of various cytokines/chemokines due to activated Th(2) immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide in the context of particular base sequence (CpG motifs) are known to have the immunostimulatory activities in mice and to convert from Th(2) to Th(1) immune responses in AD. We aimed to investigate that CpG ODN, especially phosphodiester form, can stimulate the protective immunity in NC/Nga mice with AD. We isolated BMDCs from NC/Nga mice and then, cultured with GM-CSF and IL-4 for 6 days, and treated for 2 days by either phosphorothioate ODN or phosphodiester ODN. CpG ODN-treated DCs resulted in more production of IL-12. When CpG ODN-treated DCs were intravenously injected into the NC/Nga mice, the NC/Nga mice with CpG ODN-treated DCs showed significant improvement of AD symptoms and decrease of IgE level. Histopathologically, the NC/Nga mice skin with CpG ODN-treated DCs showed the decreased IL-4 and TARC expression comparing with non-injected mice. These results may suggest that phosphodiester CpG ODN-treated DCs might function as a potent adjuvant for AD in a mouse model.     

Inability of IL-12 to down-regulate IgE synthesis due to defective production of IFN-gamma in atopic NC/Nga mice.

NC/Nga mice raised in nonsterile circumstances spontaneously suffer from atopic dermatitis-like skin lesions with IgE hyperproduction. We investigated effects of rIL-12 on the IgE production in NC/Nga mice. rIL-12 administration was successful to suppress the increase of IgE levels in BALB/c mice immunized with OVA and aluminum hydroxide, but failed to abrogate that in NC/Nga mice. Both in vivo and in vitro IFN-gamma production induced by rIL-12 was less in NC/Nga mice than in BALB/c mice. Addition of rIFN-gamma to rIL-4 and LPS completely abrogated IgE production by B cells of BALB/c mice, but was insufficient to suppress it by B cells of NC/Nga mice. In splenic cells pretreated with Con A, STAT4 was phosphorylated at the tyrosine residue by addition of rIL-12, which was more weakly inducible in NC/Nga mice than in BALB/c mice. Finally, we examined the preventive ability of rIL-12 on the clinical aspects of atopic dermatitis in NC/Nga mice. rIL-12 administration resulted in exacerbation of development of the skin lesions and IgE production in NC/Nga mice raised in nonsterile circumstances. These results suggest that defective production of IFN-gamma by T cells less sensitive to IL-12 and low responsiveness of B cells to IFN-gamma may contribute to IgE hyperproduction in NC/Nga mice, and that IL-12 may have no ability to improve the clinical aspects of NC/Nga mice.     

Deletion polymorphisms in the promoter region of Fcgamma receptor IIB is not associated with antigen-specific IgG2a and IgG2b antibody responses in NC/Nga mice

Fcgamma receptor (R) IIB, a low-affinity FcR for IgG, inhibits B cell Ag R (BCR)-mediated activation when these two receptors are cross-linked by Ag and IgG-containing immune complexs (ICs). We found deletion polymorphisms in the promoter region of fcgr2b in NC/Nga mice, a model for human atopic dermatitis. NC/Nga mice produced significantly higher levels of ovalbumin (OVA)-specific IgG, IgG2a and IgG2b than did BALB/c mice. Analysis of (BALB/c x NC/Nga)F1 x BALB/c or (BALB/c x NC/Nga) F1 x NC/Nga backcross mice revealed that deletion polymorphisms of fcgr2b in NC/Nga mice does not directly regulate hyper OVA-specific IgG2a and IgG2b Ab responses.     

Reactive nitrogen species formation in eosinophils and imbalance in nitric oxide metabolism are involved in atopic dermatitis-like skin lesions in NC/Nga mice

Nitric oxide (NO) and reactive nitrogen species (RNS) have been implicated in the pathogenesis of inflammatory diseases. However, the involvement of NO and RNS in atopic dermatitis (AD), a pruritic inflammatory skin diseases, is not fully understood. In this study, we investigated the contribution of NO and RNS to the development of AD-like skin lesions in NC/Nga mice, an animal model for human AD. AD-like skin lesions were observed in NC/Nga mice kept under conventional conditions but not in specific pathogen-free conditions. The expression of inducible NO synthase (iNOS) and endothelial NOS (eNOS) proteins was upregulated in the dermal lesions, and that of neuronal NOS (nNOS) was downregulated in the epidermal lesions of the skin. Although the concentrations of NO2(-) and NO3(-) were lower, protein-bound nitrotyrosine content was significantly increased in the skin lesions. Immunohistochemical localization of nitrotyrosine was observed in almost all eosinophils. These results suggest that RNS formation in eosinophils and imbalance of NO metabolism are involved in the pathogenesis of AD-like skin lesions in NC/Nga mice.     

DNCB致敏/激发Nc/Nga小鼠特应性皮炎模型的建立

目的研究外用2,4-二硝基氯苯（DNCB）对Nc/Nga小鼠的致敏作用,探索建立特应性皮炎（AD）模型的方法。方法外用1%DNCB7周,间隔为1周,重复刺激并致敏7周大NC/Nga小鼠的双侧耳朵及背部皮肤。结果外用DNCB7周可以引起显著的炎症,并伴有高滴度的IgE和IL-4,利用HE染色进行组织病理分析,提示表皮炎性细胞明显增加。结论外用DNCB重复刺激Nc/Nga小鼠能产生AD样皮炎,可以作为研究AD病因及治疗AD的有效动物模型。     

Development of Eczema Vaccinatum in Atopic Mouse Models and Efficacy of MVA Vaccination against Lethal Poxviral Infection

Smallpox vaccine based on live, replicating vaccinia virus (VACV) is associated with several potentially serious and deadly complications. Consequently, a new generation of vaccine based on non-replicating Modified vaccinia virus Ankara (MVA) has been under clinical development. MVA seems to induce good immune responses in blood tests, but it is impossible to test its efficacy in vivo in human. One of the serious complications of the replicating vaccine is eczema vaccinatum (EV) occurring in individuals with atopic dermatitis (AD), thus excluding them from all preventive vaccination schemes. In this study, we first characterized and compared development of eczema vaccinatum in different mouse strains. Nc/Nga, Balb/c and C57Bl/6J mice were epicutaneously sensitized with ovalbumin (OVA) or saline control to induce signs of atopic dermatitis and subsequently trans-dermally (t.d.) immunized with VACV strain Western Reserve (WR). Large primary lesions occurred in both mock- and OVA-sensitized Nc/Nga mice, while they remained small in Balb/c and C57Bl/6J mice. Satellite lesions developed in both mock- and OVA-sensitized Nc/Nga and in OVA-sensitized Balb/c mice with the rate 40–50%. Presence of mastocytes and eosinophils was the highest in Nc/Nga mice. Consequently, we have chosen Nc/Nga mice as a model of AD/EV and tested efficacy of MVA and Dryvax vaccinations against a lethal intra-nasal (i.n.) challenge with WR, the surrogate of smallpox. Inoculation of MVA intra-muscularly (i.m.) or t.d. resulted in no lesions, while inoculation of Dryvax t.d. yielded large primary and many satellite lesions similar to WR. Eighty three and 92% of mice vaccinated with a single dose of MVA i.m. or t.d., respectively, survived a lethal i.n. challenge with WR without any serious illness, while all Dryvax-vaccinated animals survived. This is the first formal prove of protective immunity against a lethal poxvirus challenge induced by vaccination with MVA in an atopic organism.     

Efficient Immuno-Modulation of TH1/TH2 Biomarkers in 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis: Nanocarrier-Mediated Transcutaneous Co-Delivery of Anti-Inflammatory and Antioxidant Drugs

The present study was conducted with the aim to investigate the immuno-modulatory and histological stabilization effects of nanocarrier–based transcutaneous co-delivery of hydrocortisone (HC) and hydroxytyrosol (HT). In this investigation, the clinical and pharmacological efficacies of nanoparticle (NP)-based formulation to alleviate 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis (AD) was explored by using an NC/Nga mouse model. Ex vivo visual examination of AD induction in experimental mice indicated remarkable control of NP-based formulations in reducing pathological severity of AD-like skin lesions. Therapeutic effectiveness of NP-based formulations was also evaluated by comparing skin thickness of AD-induced NP-treated mice (456±27 µm) with that of atopic mice (916±37 µm). Analysis of the immuno-spectrum of AD also revealed the dominance of NP-based formulations in restraining immunoglobulin-E (IgE), histamine, prostaglandin-E₂ (PGE₂), vascular endothelial growth factor-α (VEGF-α), and T-helper cells (T_H1/T_H2) producing cytokines in serum and skin biopsies of tested mice. These anti-AD data were further supported by histological findings that revealed alleviated pathological features, including collagen fiber deposition, fibroblasts infiltration, and fragmentation of elastic fibers in experimental mice. Thus, NP-mediated transcutaneous co-delivery of HC and HT can be considered as a promising therapy for managing immunological and histological spectra associated with AD.     

Inhibitory Effects of Heartwood Extracts of Broussonetia kazinoki Sieb on the Development of Atopic Dermatitis in NC/Nga Mice

We investigated the effects of a topically applied extract of the heartwood of Broussonetia kazinoki Sieb (B. kazinoki) on atopic dermatitis (AD)-like skin lesions induced by an extract of the house-dust mite Dermatophagoides farina in NC/Nga mice. We found that topically applied B. kazinoki extract suppressed the histological manifestations of AD-like skin lesions, and decreased the levels of plasma immunoglobulin E (IgE) and interleukin-4 (IL-4) in the mice. Moreover, B. kazinoki inhibited the induction of thymus-and-activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), and regulated-on-activation-normal T cell-expressed-and-secreted chemokine (RANTES/CCL5) in HaCaT cells activated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In conclusion, our results suggest that B. kazinoki extract has therapeutic advantages in the treatment of AD.     

Genuine traditional Korean medicine,Naju Jjok (Chung-Dae,Polygonum tinctorium) improves 2,4-dinitrofluorobenzene-induced atopic dermatitis-like lesional skin

PurposeNaju Jjok (NJJ, Polygonum tinctorium) is a clear heat and release toxin medicinal. It has been used to treat various inflammatory diseases and as a dye in clothing in traditional Korean medicine. However, the effect of NJJ on atopic dermatitis (AD) has not been elucidated. Therefore, we examined whether NJJ would have an inhibitory effect on AD using the mimic AD murine model and in vitro model.MethodsWe treated NJJ on 2,4-dinitrofluorobenzene (DNFB)-induced AD-like skin lesions in NC/Nga mice, phorbol myristate acetate/calcium ionophore A23187-stimulated human mast cell line (HMC-1) cells, and anti-CD3/anti-CD28-stimulated splenocytes. Histological analysis, ELISA, PCR, and Western blot analysis were performed.ResultsThe oral administration with NJJ suppressed the total clinical severity in DNFB-induced AD-like lesional skin. NJJ significantly suppressed the levels of inflammatory mRNA and protein in AD-like lesional skin. NJJ significantly suppressed the levels of IgE and interleukin-4 in the serum of DNFB-induced AD mice. The expression of mast cells-derived caspase-1 was suppressed by NJJ in AD-like lesional skin. In addition, topical application with NJJ improved clinical symptoms in DNFB-induced AD mice. The topical application with NJJ significantly suppressed the levels of IgE and histamine in the serum of DNFB-induced AD mice. NJJ suppressed the production and mRNA expression of TSLP by blockade of caspase-1 signal pathway in the activated HMC-1 cells. Furthermore, NJJ significantly decreased the production of tumor necrosis factor-α from the stimulated splenocytes.ConclusionsIn conclusion, these results propose curative potential of natural dye, NJJ by showing the scientific evidence on anti-AD effect of NJJ which has been used traditionally.     

Development of atopic dermatitis-like skin lesions in STAT6-deficient NC/Nga mice

Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by elevation of plasma levels of total IgE, infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 T cells. However, the role of Th2 cells in the pathogenesis of AD is not fully understood. In this study we examined the NC/Nga (NC) mouse model of AD and established STAT6-deficient (SATA6(-/-)) NC mice to investigate the relevance of IL-4-mediated immune responses. Surprisingly, these mice elicited AD-like skin lesions at equivalent frequency and time of onset compared with normal NC littermates. Histological features of the lesion in STAT6(-/-) NC mice fulfilled the criteria for the pathogenesis of AD, although these mice fail to produce IgE and Th2 cytokines. The lymph nodes proximal to the regions of skin that developed lesions exhibited massive enlargement elicited by the accumulation of activated IFN-gamma-secreting T cells. Moreover, caspase I, IL-18, IL-12, and IFN-gamma are found to be highly expressed at the skin lesion, occurring simultaneously with elevation of eotaxin 2 and CCR3 expression. Therefore, the Th2-mediated immune response is not necessary for the development of AD-like skin disease in NC mice. The skin microenvironment that favored IFN-gamma production tightly correlates with the skin disease in NC mice through the infiltration of eosinophils.     

The inhibitory effect of naringenin on atopic dermatitis induced by DNFB in NC/Nga mice

Aims

Atopic dermatitis (AD) is a chronic and relapsing inflammatory dermatitis characterized by pruritic and eczematous skin lesions. Here, we investigated the therapeutic effect of the fruit flavonoid naringenin on DNFB induced atopic dermatitis mice model.

Main methods

AD-like skin lesion was induced by repetitive skin contact with DNFB in NC/Nga mice and the effects of the fruit flavonoid naringenin were evaluated on the basis of histopathological findings of skin, ear swelling and cytokine production of CD4⁺T cells.

Key findings

Intraperitoneal injection of naringenin for one week after DNFB challenge significantly lowered ear swelling and improved back skin lesions. In addition, naringenin significantly suppressed production of interferon-gamma (IFN-γ) by activated CD4⁺ T cells and serum IgE level. Furthermore, naringenin reduced DNFB-induced infiltration of eosinophils, mast cells, CD4⁺ T cells, and CD8⁺ T cells in skin lesions.

Significance

Naringenin may suppress the development of AD-like skin lesions in DNFB-treated NC/Nga mice by reducing IFN-γ production of activated CD4⁺ T cells, serum IgE levels and infiltration of immune cells to skin lesion.     

Low susceptibility of NC/Nga mice to the lipopolysaccharide-mediated lethality with D-galactosamine sensitization and the involvement of fewer natural killer T cells

The LPS-mediated lethality of NC/Nga mice, having fewer NKT cells, was examined by using d-galactosamine (d-GalN)-sensitization. The NC/Nga mice were not killed by a simultaneous administration of d-GalN and LPS whereas all C57BL/6 (B6) control mice were killed. The injection of d-GalN and LPS failed to elevate the levels of serum alanine aminotransferase and caspase 3 in the liver tissues of NC/Nga mice. Further, the nitric oxide (NO) level of the d-GalN- and LPS-injected NC/Nga mice was much lower than those of the B6 mice. The expression of an inducible NO synthase (iNOS) was significantly reduced in the livers of NC/Nga mice. However, there was no significant difference in LPS-induced TNF-α production between B6 mice and NC/Nga mice. The NC/Nga mice had an impaired expression of IFN-γ protein and mRNA in response to d-GalN and LPS. The pretreatment with α-galactosylceramide (α-GalCer), which activates Vα14(+) NKT cells and induces the production of IFN-γ, rendered NC/Nga mice more susceptible to the LPS-mediated lethality. The livers of NC/Nga mice had fewer NKT cells compared to B6 mice. Taken together, it is suggested that the resistance of NC/Nga mice to the LPS-mediated lethality with d-GalN sensitization depended on the impaired IFN-γ production caused by fewer NKT cells and reduced NO production that followed.     

A Comprehensive Behavioral Test Battery to Assess Learning and Memory in 129S6/Tg2576 Mice

Transgenic Tg2576 mice overexpressing human amyloid precursor protein (hAPP) are a widely used Alzheimer’s disease (AD) mouse model to evaluate treatment effects on amyloid beta (Aβ) pathology and cognition. Tg2576 mice on a B6;SJL background strain carry a recessive rd1 mutation that leads to early retinal degeneration and visual impairment in homozygous carriers. This can impair performance in behavioral tests that rely on visual cues, and thus, affect study results. Therefore, B6;SJL/Tg2576 mice were systematically backcrossed with 129S6/SvEvTac mice resulting in 129S6/Tg2576 mice that lack the rd1 mutation. 129S6/Tg2576 mice do not develop retinal degeneration but still show Aβ accumulation in the brain that is comparable to the original B6;SJL/Tg2576 mouse. However, comprehensive studies on cognitive decline in 129S6/Tg2576 mice are limited. In this study, we used two dementia mouse models on a 129S6 background—scopolamine-treated 129S6/SvEvTac mice (3–5 month-old) and transgenic 129S6/Tg2576 mice (11–13 month-old)–to establish a behavioral test battery for assessing learning and memory. The test battery consisted of five tests to evaluate different aspects of cognitive impairment: a Y-Maze forced alternation task, a novel object recognition test, the Morris water maze, the radial arm water maze, and a Y-maze spontaneous alternation task. We first established this behavioral test battery with the scopolamine-induced dementia model using 129S6/SvEvTac mice and then evaluated 129S6/Tg2576 mice using the same testing protocol. Both models showed distinctive patterns of cognitive impairment. Together, the non-invasive behavioral test battery presented here allows detecting cognitive impairment in scopolamine-treated 129S6/SvEvTac mice and in transgenic 129S6/Tg2576 mice. Due to the modular nature of this test battery, more behavioral tests, e.g. invasive assays to gain additional cognitive information, can easily be added.     

Modulation of scratching behavior by silencing an endogenous cyclooxygenase-1 gene in the skin through the administration of siRNA

BACKGROUND: RNA interference (RNAi) is rapidly becoming a major tool that is revolutionizing research in the bioscience and biomedical fields. To apply the RNAi technique in vivo, it is crucial to develop appropriate methods of guiding the short interfering RNA (siRNA) molecules to the right tissues and cells. Here, we demonstrate an efficient method for performing gene knockdown in the body skin using the in vivo electro-transduction of siRNA. Using this method, we examined whether the targeted silencing of the cyclooxygenase (COX) gene in the skin could modulate the scratching behavior of an atopic dermatitis mouse model. METHODS: NC/Nga mice were used as the atopic dermatitis model. Using our optimized in vivo electroporation conditions, siRNAs were introduced into the skin; the silencing efficiency was then analyzed by Western blotting, measuring the levels of prostaglandins, and immunohistochemistry. The scratching behaviors of the mice were measured using an automatic system. RESULTS: Targeted silencing of the COX-1 gene using our in vivo siRNA technique significantly accelerated the scratching behavior of NC/Nga mice, whereas the COX-2 siRNA showed no effect. In addition, the effect of COX-1 siRNA was mimicked by treatment with a COX-1-selective inhibitor (SC-560). CONCLUSIONS: We have demonstrated the successful silencing of endogenous gene expression in the skin using the intradermal transfection of unmodified siRNA via electroporation. Using this method, we revealed that COX- 1-mediated prostaglandins may act as endogenous inhibitors of scratching behavior.