Auricular Nodes Research Articles

Sub-acute oral exposure to lowest observed adverse effect level of nivalenol exacerbates atopic dermatitis in mice via direct activation of mitogen-activated protein kinase signal in antigen-presenting cells.

This study investigated the immunotoxic effects of the mycotoxin nivalenol (NIV) using antigen-presenting cells and a mouse model of atopic dermatitis (AD). In vitro experiments were conducted using a mouse macrophage cell line (RAW 264.7) and mouse dendritic cell line (DC 2.4). After cells were exposed to NIV (0.19-5µmol) for 24h, the production of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) was quantified. To further investigate the inflammatory cytokine production pathway, the possible involvement of mitogen-activated protein kinase (MAPK) pathways, such as ERK1/2, p-38, and JNK, in NIV exposure was analyzed using MAPK inhibitors and phosphorylation analyses. In addition, the pro-inflammatory effects of oral exposure to NIV at low concentrations (1 or 5ppm) were evaluated in an NC/Nga mouse model of hapten-induced AD. In vitro experiments demonstrated that exposure to NIV significantly enhanced the production of TNFα. In addition, it also directly induced the phosphorylation of MAPK, indicated by the inhibition of TNFα production following pretreatment with MAPK inhibitors. Oral exposure to NIV significantly exacerbated the symptoms of AD, including a significant increase in helper T cells and IgE-produced B cells in auricular lymph nodes and secretion of pro-inflammatory cytokines, such as IL-4, IL-5, and IL-13, compared with the vehicle control group. Our findings indicate that exposure to NIV directly enhanced the phosphorylation of ERK1/2, p-38, and JNK, resulting in a significant increase in TNFα production in antigen-presenting cells, which is closely related to the development of atopic dermatitis.

Lymphatic drainage patterns of malignant skin tumors in the head and neck region: a single-center retrospective study.

This study aimed to clarify the relationship between primary site and lymphatic drainage pattern for malignant skin tumors in the head and neck region. Malignant melanoma and squamous cell carcinoma in the head and neck region are known to have poor prognosis because of lymph node metastasis. Nevertheless, numerous aspects of lymphatic drainage patterns remain elusive. We statistically analyzed data of 47 patients with malignant skin tumors in the head and neck region. Information was collected on the patients' clinical characteristics, primary tumor site, and lymphatic drainage patterns. The parotid lymph nodes drained the greatest amount of lymph from skin tumors of the head and neck. Important lymphatic drainage pathways were the superficial cervical nodes for primary tumors in the buccal/nasal region, level IA and level IB nodes for primary tumors in the lip region, the occipital nodes, posterior auricular nodes, and level VA nodes in the parietal/occipital region, and the preauricular nodes in the auricular region. These findings have considerable significance in terms of understanding lymphatic drainage patterns for malignant skin tumors in the head and neck and may be useful for clinical decision-making and when planning treatment. Further research and clinical applications are expected to contribute to an improved prognosis in patients with cutaneous head and neck malignancies.

Sebaceous cell carcinoma of the scalp in a young male: A rare case report

Sebaceous carcinoma is a rare locally aggressive neoplasm of the sebaceous gland, which occurs frequently in elderly males. It constitutes 0.2 to 4.6% of all the malignant cutaneous tumours and occurs commonly in the periocular area. Scalp is a rare site for this tumor. Here we report a case of sebaceous carcinoma in a young 28 years old male patient who presented with firm to hard exophytic growth over right posterior occipital region with enlarged suboccipital and post auricular lymph nodes. Wide local excision was done and a diagnosis of sebaceous carcinoma of the scalp with lymph node metastasis was rendered after histopathological and Immunohistochemical examination.

Combination Therapy with Betamethasone and Josamycin Demonstrates Superior Therapeutic Efficacy in an NC/Nga Mouse Model of Atopic Dermatitis.

We have previously demonstrated the excellent bactericidal activity of josamycin against Staphylococcus aureus isolated from patients with atopic dermatitis (AD), with therapeutic efficacy equal to that of betamethasone. The present study was designed to evaluate the effectiveness of combination therapy with betamethasone and josamycin for AD. Betametasone (0.1%) and josamycin (0.1%) were topically administered to NC/Nga mice with severe AD-like skin lesions. Skin severity scores, histological changes in skin lesions, and serum immunoglobulin E (IgE) levels were assessed as indicators of therapeutic efficacy. Topical treatment with both drugs suppressed the skin severity score to a greater degree than betamethasone alone. This was associated with a reduction of epidermal thickening, a reduced density of dermal cellular infiltration, a decreased mast cell count in the dermis, and a reduced serum IgE level. In addition, both drugs in combination markedly reduced the expression of interferon (IFN)-γ and interleukin (IL)-4 in auricular lymph node cells, as well as the S. aureus count on the lesioned skin. These results show that simultaneous topical application of both drugs can ameliorate severe AD-like skin lesions in NC/Nga mice. It is suggested that combination therapy with betamethasone and josamycin would be beneficial for control of severe AD lesions colonized by S. aureus by inhibiting the development of both T helper (Th) type 1 (Th1) and Th2 cells and also through elimination of superficially located S. aureus.

Oral exposure to citrinin significantly exacerbates the pathophysiology of a mouse model of imiquimod-induced psoriasis via direct activation of dendritic cell.

Citrinin, a mycotoxin produced by Penicillium citrinum and Penicillium verrucosum, mainly contaminates cereals. The aim of study was to investigate the novel immunoreactive effect of citrinin using a mouse model of psoriasis. A mouse model of psoriasis was generated by topical application of 5% imiquimod in female BALB/c mice. Standard rodent diet and rice samples with 3ppm of citrinin were mixed to obtain a final citrinin concentration of 0.3ppm, and a citrinin-contaminated diet was fed to mice daily. Skin thickness, scratching behavior, and trans epidermal water loss (TEWL) were monitored continuously during the imiquimod application. Immediately after the final imiquimod application, ear skin and auricular lymph node (LN) were sampled for further analysis. Only a slight increase was observed in skin thickness in the citrinin exposure group; however, citrinin exposure significantly exacerbated hyperkeratinization and inflammatory cell infiltration in histological evaluation. TEWL, which is representative of cutaneous barrier function, was significantly increased by citrinin exposure. In terms of immune function, the number of immune cells in LN (T cells and dendritic cells) and gene expression of interleukin (IL)-17 in skin tissue were significantly increased by citrinin exposure. Direct interaction of dendritic cells (DCs) in citrinin-induced psoriasis development was further examined by proinflammatory cytokine determination in THP-1 cells and murine bone marrow derived DCs. IL-6 and/or tumor necrosis factor α were significantly increased by citrinin exposure. Taken together, our results imply that oral exposure to citrinin exacerbates the symptoms of a mouse model of psoriasis via direct activation of DCs.

Effects of dual therapy with betamethasone and tetracycline in a NC/Nga mouse model of atopic dermatitis.

Purpose: Our previous study has demonstrated that tetracycline exerts excellent bactericidal activity against Staphylococcus aureus strains isolated from patients with atopic dermatitis (AD) while simultaneously inhibiting the development of T helper (Th) type 2 (Th2) cells. The present study was designed to evaluate the effectiveness of dual therapy with betamethasone and tetracycline for AD. Methods: Betametasone (0.1%) and tetracycline (3%) were topically administered to NC/Nga mice with AD-like skin lesions. Skin severity scores, histological changes to the lesioned skin, and serum IgE levels were assessed as indicators of therapeutic effectiveness. Results: Topical treatment with both drugs reduced the skin severity score more significantly than was the case with betamethasone alone or tetracycline alone. This was associated with a reduction in the degree of epidermal thickening, the density of cellular infiltration into the dermis, the mast cell count in the dermis and the serum IgE concentration. Furthermore, the degree of Th1/Th2 cell development in auricular lymph nodes and the S. aureus count on the lesioned skin were synergistically suppressed by simultaneous application of both drugs. Conclusion: The present results show that simultaneous topical application of betamethasone and tetracycline synergistically ameliorates AD-like skin lesions in NC/Nga mice. This suggests that dual therapy with betamethasone and tetracycline for AD lesions colonized by S. aureus might be one of the best options for inhibiting the development of both Th1 and Th2 cells and acting on superficially located S. aureus .

Assessment of skin sensitization properties of few-layer graphene and graphene oxide through the Local Lymph Node Assay (OECD TG 442B)

Skin contact is one of the most common exposure routes to graphene-based materials (GBMs) during their small-scale and industrial production or their use in technological applications. Nevertheless, toxic effects in humans by cutaneous exposure to GBMs remain largely unexplored, despite skin contact to other related materials has been associated with adverse effects. Hence, this in vivo study was carried out to evaluate the cutaneous effects of two GBMs, focusing on skin sensitization as a possible adverse outcome.Skin sensitization by few-layer graphene (FLG) and graphene oxide (GO) was evaluated following the Organization for Economic Cooperation and Development (OECD) guideline 442B (Local Lymph Node Assay; LLNA) measuring the proliferation of auricular lymph node cells during the induction phase of skin sensitization. Groups of four female CBA/JN mice (8–12 weeks) were daily exposed to FLG or GO through the dorsal skin of each ear (0.4–40 mg/mL, equal to 0.01–1.00 mg/ear) for 3 consecutive days, and proliferation of auricular lymph node cells was evaluated 3 days after the last treatment. During this period, no clinical signs of toxicity and no alterations in body weight and food or water consumptions were observed. In addition, no ear erythema or edema were recorded as signs of irritation or inflammation. Bromo-deoxyuridine (BrdU) incorporation in proliferating lymphocytes from ear lymph nodes (stimulation indexes <1.6) and the histological analysis of ear tissues excluded sensitizing or irritant properties of these materials, while myeloperoxidase activity in ear biopsies confirmed no inflammatory cells infiltrate. On the whole, this study indicates the absence of sensitization and irritant potential of FLG and GO.

Observation of hapten-induced sensitization responses for the development of a mouse skin sensitization test, including the elicitation phase

The only official method that can detect the skin sensitizing potential of chemicals, including the elicitation response, is the OECD test guideline (TG) 406. However, this guideline uses guinea pigs, which requires complex procedures. Since a simple and complete test method for evaluating skin sensitization is needed, especially for mechanistic studies of skin sensitization, this study confirmed the reactivity of mice to skin sensitizing substances. We set up a protocol involving one induction exposure of the test substance to the back skin, followed by three challenge exposures to the auricle (Protocol 2), and compared their skin sensitization responses with the results of two exposures to the auricle and back skin every 2 weeks (Protocol 1) and a local lymph node assay (TG442B). A hapten 2,4-dinitrofluorobenzene caused significant auricular thickening, skin inflammation, and enlarged auricular lymph nodes in Protocols 1 and 2. These changes were more pronounced in Protocol 2. Plasma IgE and IgG1 and gene expression of IL4, IFNγ, and perforin were significantly increased in Protocol 2. Cell proliferation in the auricular lymph nodes was observed in both protocols as in TG442B. These results indicate that Protocol 2 can be a good candidate for a relatively simple skin sensitization test.

A CASE REPORT AND LITERATURE REVIEW: PRIMARY CARCINOMA OF LACRIMAL GLAND TREATED WITH ADJUVANT CHEMO- RADIATION.

Primary Sebaceous Carcinoma of Lacrimal Gland, possibly arising from heterotopic sebaceous tissue, is extremely rare and must be differentiated from secondary invasion of the Orbit by a primary Eyelid tumor/metastatic spread from other areas of the body. It is known to be aggressive So, delay in diagnosis can increase the chance of local recurrence and metastasis. In this article we report a case of Primary Sebaceous Carcinoma of the lacrimal gland, who underwent surgery with positive margin received Adjuvant radiotherapy in our Institute. A 55-year-OldCASE REPORT: Lady presented with Left eyelid swelling. MRI ORBITS &amp; PNS reveals malignant mass of approx. 4.3 X 3.8 cm size in superior &amp; temporal aspect of the left Orbit arising from the left upper eyelid compressing left globe and inseparable from the left lacrimal gland with approx. 2.3X 1.5 cm sized enlarged left Pre- auricular Node. No optic nerve encasement, Orbital wall Destruction &amp; Extension to adjacent paranasal sinuses is seen. CECT Thorax &amp; Whole abdomen – Normal. FNAC from eyelid swelling &amp; Pre- Auricular Node S/o Sebaceous Carcinoma with metastasis. Patient underwent left Orbital Exenteration + left supercial parotid split skin graft. HPE show the tumor mass of size 4.5 X 4X 3 cm, completely replacing tarsal plate of eyelid &amp; tumor mass show a highly inltrative malignant tumor with morphology consistent with sebaceous carcinoma with positive margin and tissue from left parotid show inltration by the sebaceous carcinoma (Possibly metastasis to the Intra-parotid node with extra nodal extension). Patient received Adjuvant/post op Chemoradiation 60 Gy/30#/ 6weeks/Rapid Arc technique with weekly 6 cycles chemotherapy Inj. Cisplatin. Patient requires close monitoring for any sign of recurrence / Distant metastasis. A Rare case report of sebaceousCONCLUSION: carcinoma of the Lacrimal Gland which requires appropriate evaluation &amp; management for better prognosis.

Rubella, German Measles

Rubella is caused by an RNA virus. Infection results mostly in few or no symptoms. Viremia and viral shedding start before a rash may be seen. German physicians were probably the first to describe rubella in the early 19th century, hence the name "German measles". A British physician reported an outbreak in a boys’ school in India in 1841. He used the word rubella, "little red", a Latin diminutive from ruber (red). Rubella is often indistinguishable from other viral exanthematous diseases, but palpable posterior auricular and suboccipital lymph nodes are almost pathognomonic. Rubella infection during pregnancy may result in cataract, heart disease and deafness in an infant, forming the key triad defining “congenital rubella syndrome”, CRS. Also, mental retardation is common. After birth, rubella is a mild disease with rare complications only. There is no treatment for rubella or CRS, but vaccination programs usually with MMR-vaccine maintaining high vaccine uptake over time can virtually eliminate rubella.

Differential Early in vivo Dynamics and Functionality of Recruited Polymorphonuclear Neutrophils After Infection by Planktonic or Biofilm Staphylococcus aureus.

Staphylococcus aureus is a human pathogen known for its capacity to shift between the planktonic and biofilm lifestyles. In vivo, the antimicrobial immune response is characterized by the recruitment of inflammatory phagocytes, namely polymorphonuclear neutrophils (PMNs) and monocytes/macrophages. Immune responses to planktonic bacteria have been extensively studied, but many questions remain about how biofilms can modulate inflammatory responses and cause recurrent infections in live vertebrates. Thus, the use of biologically sound experimental models is essential to study the specific immune signatures elicited by biofilms. Here, a mouse ear pinna model of infection was used to compare early innate immune responses toward S. aureus planktonic or biofilm bacteria. Flow cytometry and cytokine assays were carried out to study the inflammatory responses in infected tissues. These data were complemented with intravital confocal imaging analyses, allowing the real-time observation of the dynamic interactions between EGFP + phagocytes and bacteria in the ear pinna tissue of LysM-EGFP transgenic mice. Both bacterial forms induced an early and considerable recruitment of phagocytes in the ear tissue, associated with a predominantly pro-inflammatory cytokine profile. The inflammatory response was mostly composed of PMNs in the skin and the auricular lymph node. However, the kinetics of PMN recruitment were different between the 2 forms in the first 2 days post-infection (pi). Two hours pi, biofilm inocula recruited more PMNs than planktonic bacteria, but with decreased motility parameters and capacity to emit pseudopods. Inversely, biofilm inocula recruited less PMNs 2 days pi, but with an “over-activated” status, illustrated by an increased phagocytic activity, CD11b level of expression and ROS production. Thus, the mouse ear pinna model allowed us to reveal specific differences in the dynamics of recruitment and functional properties of phagocytes against biofilms. These differences would influence the specific adaptive immune responses to biofilms elicited in the lymphoid tissues.

Gomisin M2 Ameliorates Atopic Dermatitis-like Skin Lesions via Inhibition of STAT1 and NF-κB Activation in 2,4-Dinitrochlorobenzene/Dermatophagoides farinae Extract-Induced BALB/c Mice.

The extracts of Schisandra chinensis (Turcz.) Baill. (Schisandraceae) have various therapeutic effects, including inflammation and allergy. In this study, gomisin M2 (GM2) was isolated from S. chinensis and its beneficial effects were assessed against atopic dermatitis (AD). We evaluated the therapeutic effects of GM2 on 2,4-dinitrochlorobenzene (DNCB) and Dermatophagoides farinae extract (DFE)-induced AD-like skin lesions with BALB/c mice ears and within the tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated keratinocytes. The oral administration of GM2 resulted in reduced epidermal and dermal thickness, infiltration of tissue eosinophils, mast cells, and helper T cells in AD-like lesions. GM2 suppressed the expression of IL-1β, IL-4, IL-5, IL-6, IL-12a, and TSLP in ear tissue and the expression of IFN-γ, IL-4, and IL-17A in auricular lymph nodes. GM2 also inhibited STAT1 and NF-κB phosphorylation in DNCB/DFE-induced AD-like lesions. The oral administration of GM2 reduced levels of IgE (DFE-specific and total) and IgG2a in the mice sera, as well as protein levels of IL-4, IL-6, and TSLP in ear tissues. In TNF-α/IFN-γ-stimulated keratinocytes, GM2 significantly inhibited IL-1β, IL-6, CXCL8, and CCL22 through the suppression of STAT1 phosphorylation and the nuclear translocation of NF-κB. Taken together, these results indicate that GM2 is a biologically active compound that exhibits inhibitory effects on skin inflammation and suggests that GM2 might serve as a remedy in inflammatory skin diseases, specifically on AD.

Topical treatment with mastic (resin from Pistacia lentiscus) elicits anti-inflammatory and anti-pruritic responses by modulating keratinocyte activation in a mouse model of allergic dermatitis

BackgroundAs the number of patients with skin allergies, including atopic dermatitis, has increased rapidly, therapeutic options such as anti-IL-31 antibody and Janus kinase inhibitor have been developed recently. However, many concerns remain regarding the adverse effects and cost of these drugs; therefore, development of supplements that could support the effect of therapeutic agents is always required. PurposeThe aim of this study was to develop preventive and supportive options for skin allergies by focusing on a natural product called “Mastic”. Methods: Initially, the anti-inflammatory and anti-pruritic responses of 3% and 30% Mastic topical treatment were investigated in a mouse model of allergic contact dermatitis, generated by topical application of toluene-2,4-diisocyanate (TDI), a hapten that induces type 2 helper T cells. After itch behaviour and ear-swelling response were monitored, serum, auricular lymph nodes, and skin tissues were collected to analyse immunocyte differentiation, cytokine determination, and histological changes. ResultsOur findings indicated that topical treatment with mastic significantly ameliorated ear swelling, itch behaviour, immunocyte infiltration, and cytokine production. Histological evaluation confirmed the occurrence of anti-inflammatory responses. The anti-inflammatory and anti-pruritic effects of topical treatment with mastic (3% and 5%) were further confirmed in a mouse model of atopic dermatitis which was generated by topical application of TDI in NC/Nga mice. Thickness of the back skin, AD score, transepidermal water loss (TEWL), and itch behaviour were measured weekly, and immunocyte differentiation, cytokine determination, and histological changes were also analysed. Mastic treatment significantly attenuated the skin thickness, AD score, TEWL, and itch behaviour. Corroborated reduction was observed in the numbers of T cells and IgE-B cells, as well as in pro-inflammatory cytokine production. The reproducibility of the effects of mastic was confirmed with 1% mastic ointment in a setting similar to the AD mouse model. In vitro evaluation of keratinocytes indicated that mastic pre-exposure induced a significant dose-dependent decrease in cytokine production. ConclusionOur findings thus demonstrate that topical treatment with mastic significantly ameliorate inflammatory and pruritic responses in a mouse model of allergic dermatitis.

Deceiving &amp; deceptive growth in the ear canal in an elderly female

Malignant neoplasms of the external auditory canal, middle and inner ear are rare. Squamous cell carcinoma (SCC) is the most common neoplasm to occur in this region. As such primary neoplasms of the external auditory canal and temporal bone are uncommon, as these structures more frequently are involved by the cutaneous squamous cell carcinoma (cSCC) of pinna or metastatic cSCC involving parotid gland or post auricular lymph nodes.

Effects of anti-allergy drugs on Th1 cell and Th2 cell development mediated by Langerhans cells

It is well known that Langerhans cells (LCs) work as the primary orchestrators in polarization towards T helper type 1 (Th1) or T helper type 2 (Th2) immune responses. In this study, we examined the effects of various anti-allergy drugs against the Th2 cell development by LCs. The expression of cell surface molecules on LCs was investigated using reverse transcriptase polymerase chain reaction. The effects of anti-allergy drugs on T-cell immunoglobulin and mucin domain-containing protein (TIM)-4 expression in LCs were examined to predict whether they would inhibit Th2 cell development. Next, mice were primed via the hind footpad with ovalbumin (OVA)-pulsed LCs that had been treated with selected anti-allergy drugs. After 5 days, the cytokine response in the popliteal lymph nodes was investigated by enzyme-linked immunosorbent assay. The therapeutic effects of a selected drug on atopic dermatitis (AD) were assessed using AD-like skin lesions of NC/Nga mice. The first-generation histamine H1 receptorantagonists, cyproheptadine and promethazine, and the second-generation histamine H1 receptor antagonists, emedastine and loratadine, were selected as candidate inhibitors of Th2 cell development. As expected, OVA peptide-pulsed LCs that had been treated with each drug and injected into the hind footpads of mice inhibited Th2 cell development, as represented by down-regulation of interleukin (IL)-4 production. Furthermore, the LCs that had been treated withemedastine also inhibited Th1 cell development, as represented by down-regulation of interferon (IFN)-g production. This additional inhibition of Th1 cell development was accompanied by suppression of CD40 expression in LCs. Therefore, the therapeutic effect of emedastine on AD was examined. Topical application of emedastine significantly suppressed the increase in the skin severity score in NC/Nga mice with AD-like skin lesions. This suppressive effect was associated with a decrease in the production of IFN-g and IL-4 in auricular lymph node cells. These results suggest that topical application of emedastine to skin lesions of patients with AD may provide clinical benefits through the inhibition of both Th1 cell and Th2 cell development mediated by LCs.

Acute and subacute oral administration of mycotoxin deoxynivalenol exacerbates the pro-inflammatory and pro-pruritic responses in a mouse model of allergic dermatitis.

Deoxynivalenol (DON) contamination in food is a public health concern; however, the effect of DON exposure on immune disorders including allergies remains unclear. The aim of this study is to elucidate the effect of oral exposure to DON on pro-inflammatory and pro-pruritic responses in a mouse model of allergic dermatitis, which was generated by topical application of toluene-2,4-diisocyanate (TDI), a hapten that induces type-2 helper T cells. To evaluate acute exposure to DON, the mice were orally administered vehicle alone, 0.1mg/kg DON, or 0.3mg/kg DON 48, 24, and 1h before the final challenge with TDI. To study subacute exposure, the mice were fed DON-contaminated rodent diet (0.3ppm) during the experimental period. After the itch behavior and ear-swelling response were monitored, the serum, auricular lymph node, and skin tissue were collected for analyzing immunocyte differentiation, cytokine determination, and histological changes. Acute oral administration of DON significantly enhanced pro-inflammatory responses including ear-swelling response, immunocyte infiltration, and cytokine productions. Histological evaluation supported the occurrence of pro-inflammatory responses. In contrast, acute DON exposure only slightly increased itch behavior. Subacute oral exposure to DON significantly up-regulated the inflammatory responses, but showed almost no effect on pruritic response. In vitro evaluation in dendritic cells and keratinocytes indicated that DON pre-exposure induced a dose-dependent significant increase in cytokine production. Our results imply that both acute and subacute exposures to DON are associated with pro-inflammatory responses in cutaneous allergy.

Estrogen receptor α activation aggravates imiquimod-induced psoriasis-like dermatitis in mice by enhancing dendritic cell interleukin-23 secretion.

Our recent study has reported that estrogen receptors (ERs) are involved in several types of allergy development. This study aims to investigate the possible relationship between ER activation and development of imiquimod-induced psoriasis-like dermatitis. A mouse model of imiquimod-induced psoriasis-like dermatitis was generated by 5 days of topical application of 5% of imiquimod cream on the back of the ear and the shaved back skin of male BALB/c mice. From the second day of applying 5% imiquimod cream, either ERα selective agonist (propylpyrazoletriol [PPT] 2.5 mg/kg) or ERβ selective agonist (diarylpropionitrile, DPN; 2.5 mg/kg) was administered orally for four consecutive days. Immediately after the final imiquimod cream application, scratching behavior was video monitored for 2 hours. The ear-swelling response was determined by comparing ear thickness before and after the final application of imiquimod cream. Twenty-four hours after the final imiquimod application, back skin tissue and auricular lymph nodes were isolated under isoflurane anesthesia. Oral administration of PPT significantly induced itch behavior and proinflammatory responses, including the levels of interleukin (IL)-17 and IL-22, whereas DPN treatment did not influence either pruritic or proinflammatory responses. In addition, IL-23 contribution by dendritic cells was identified using ER agonists on pretreated lipopolysaccharide (LPS)-stimulated murine bone marrow derived dendritic cells (BMDCs). PPT also significantly enhanced IL-23 secretion by LPS-stimulated BMDCs. Our findings indicate that the activation of ERα, but not ERβ, is directly associated with inflammatory and pruritic responses in a mouse model of the imiquimod-induced psoriasis by enhancing the secretion of IL-23 by dendritic cells.

Topical Application of Doxycycline Inhibits Th2 Cell Development Mediated by Langerhans Cells and Exerts a Therapeutic Effect on Atopic Dermatitis

Langerhans cells (LCs) polarize the immune milieu towards a T helper type (Th) 1 or Th2 immune response. We investigated the effects of selected tetracyclines on Th cells development mediated by LCs, and their implications for the treatment of atopic dermatitis (AD). Mice were primed with ovalbumin (OVA) peptide-pulsed LCs, which had been treated with each antibiotic, via the hind footpad. After 5 days, the Th1/Th2 cytokine response in the popliteal lymph nodes was investigated by enzyme-linked immunosorbent assay. The expression of cell surface molecules on LCs was investigated using reverse transcriptase polymerase chain reaction. The therapeutic effects of a selected antibiotic on AD-like skin lesions of NC/Nga mice were assessed in terms of the skin severity score, histological changes in the lesioned skin, the serum level of total IgE, and expression of Th1/Th2 cytokines in lymph nodes and skin lesions. Antibiotic-treated, OVA peptide-pulsed LCs inhibited development of Th2 cells but not Th1 cells. This was accompanied by suppression of T-cell immunoglobulin and mucin domain-containing protein (TIM)-4 expression in LCs. Doxycycline had the greatest activity against Staphylococcus aureus strains isolated from skin lesions of patients with AD, and a strong inhibitory effect on Th2 cell development. Doxycycline suppressed the increase in the skin severity score during the acute phase in NC/Nga mice similar to betamethasone. This suppressive effect was associated with a decrease in the serum IgE level and production of Th2 cytokines in auricular lymph node cells and skin lesions. Topical application of doxycycline to AD lesions would act on both superficial S. aureus colonization and epidermal LCs, thus possibly inhibiting the development of Th2 cells in vivo, with benefits for control of acute inflammation in AD.

385 Ruxolitinib cream ameliorates a preclinical model of skin dermatitis via modulation of inflammatory T-cell subsets

Allergic contact dermatitis, is driven by an overreaction of the body’s immune system to a normally harmless substance resulting in skin inflammation. Preclinically, topical administration of the T helper 2 (Th2) hapten fluorescein isothiocyanate (FITC) drives an immunogenic sensitization in mice and recall challenge on the ear triggers a localized inflammatory contact hypersensitivity. Pharmacological inhibition of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway has previously demonstrated efficacy in a Phase 2 clinical trial in patients with atopic dermatitis (AD) (NCT03011892). Ruxolitinib cream, a topical JAK1/2 inhibitor, is currently being evaluated in clinical trials for the treatment of mild-to-moderate AD (NCT03745638, NCT03538041 and NCT03745651). The experimental goal was to evaluate the dose-dependent efficacy of ruxolitinib cream as a topical monotherapy for the modulation of experimentally induced contact dermatitis in mice. Acute and chronic dermatitis was induced in Balb/C mice via FITC sensitization and subsequent recall response. Topical administration of ruxolitinib cream was compared to steroid (Betamethasone cream, 0.05%). Efficacy was quantified via inflammatory edema, immune phenotyping, tissue histology and immune pathway analysis. Topical administration of 1.5% ruxolitinib cream (q.d. and b.i.d) dose-dependently reduced inflammatory swelling and lichenification following recall FITC challenge in both acute (p<0.05) and chronic (p<0.05) contact dermatitis. Ruxolitinib efficacy was associated with a significant (p<0.01), dose-dependent, reduction in Th2 lymphocytes within the auricular (draining) lymph node). Pathway analysis revealed downregulation of inflammatory mechanisms consistent with JAK1/2 inhibition. Ruxolitinib cream was highly effective at inhibiting contact dermatitis in mice, ameliorated pro-inflammatory mechanisms and reduced skin thickening. Together, the data suggest that ruxolitinib cream may have potential as a therapeutic agent for the treatment of contact dermatitis.

Evaluation of skin sensitization potential of chemicals by local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry

The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.