Oral Administration of Sargassum horneri Improves the HDM/DNCB-Induced Atopic Dermatitis in NC/Nga Mice.

The present study investigated the protective effects of Sargassum horneri (S. horneri) ethanol extract (SHE) against atopic dermatitis (AD), known as an abnormal immune response in house dust mite (HDM)/2,4-dinitrochlorobenzene (DNCB)-stimulated NC/Nga mice. The oral administration of SHE attenuated the AD symptoms, including the skin dermatitis severity, transepidermal water loss (TEWL), and ear edema in HDM/DNCB-stimulated mice. Moreover, the histological analysis revealed that SHE improved epidermal hyperplasia and hyperkeratosis, and reduced the dermal infiltrations of mast cells and eosinophils. Moreover, SHE downregulated the expression levels of cytokines (interleukin (IL)-6, IL-10, and interferon (IFN)-γ) and chemokines (Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Eotaxin, and Thymus and activation-regulated chemokine (TARC)) by decreasing the expression levels of atopic initiators (IL-25 and IL-33) in HDM/DNCB-stimulated skin. The oral administration of SHE decreased the spleen size, reducing expression levels of AD-related cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IFN-γ, and TARC) by regulating the expressions of Tbx21 (T-bet), GATA Binding Protein 3 (GATA-3), and Signal transducer and activator of transcription 3 (STAT3). Moreover, SHE significantly attenuated the serum immunoglobulin (Ig)G1 and IgG2a levels in HDM/DNCB-stimulated mice. Collectively, these results suggest that S. horneri could be an ingredient of functional food against abnormal immune response.

Anti-inflammatory Effects of Variola Virus TNF Decoy Receptor in an Experimental Model of Contact Dermatitis.

BACKGROUND: Large DNA poxviruses encode a diverse family of secreted proteins that modulate host inflammatory and antiviral responses, in particular by inhibiting one of the key players of the mammalian immune system, the tumor necrosis factor (TNF). METHODS: We investigated the effects of a recombinant variola (smallpox) virus TNF-decoy receptor (VARV-CrmB) in a murine model of contact dermatitis. Our results demonstrate that the VARV-CrmB protein significantly reduces the 2,4-dinitrochlorbenzene (DNCB)-induced migration of skin leukocytes during the sensitization phase and suppresses ear oedema during the elicitation phase of the contact reaction. RESULTS: Studies focusing on the bone marrow hematopoiesis in the contact dermatitis model revealed that the epicutaneous co-application of DNCB and VARV-CrmB protein normalized the DNCBinduced effects to control levels. CONCLUSION: As an effective TNF antagonist, the VARV-CrmB protein might be conceived as a beneficial candidate for further research and development of therapeutic approaches in the field of the inflammatory skin diseases.

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na+/H+ exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Molecular cloning, biochemical characterization, and expression analysis of two glutathione S-transferase paralogs from the big-belly seahorse (Hippocampus abdominalis).

Glutathione S-transferases (GSTs, EC 2.5.1.18) are important Phase II detoxifying enzymes that catalyze hydrophobic, electrophilic xenobiotic substance with the conjugation of reduced glutathione (GSH). In this study, GSTµ and GSTρ paralogs of GST in the big belly seahorse (Hippocampus abdominalis; HaGSTρ, HaGSTµ) were biochemically, molecularly, functionally characterized to determine their detoxification range and protective capacities upon different pathogenic stresses. HaGSTρ and HaGSTµ are composed of coding sequences of 681bp and 654bp, which encode proteins 225 and 217 amino acids, with predicted molecular masses of 26.06kDa and 25.74kDa respectively. Sequence analysis revealed that both HaGSTs comprise the characteristic GSH-binding site in the thioredoxin-like N-terminal domain and substrate binding site in the C-terminal domain. The recombinant HaGSTρ and HaGSTµ proteins catalyzed the model GST substrate 1-chloro-2, 4-dinitrobenzene (CDNB). Enzyme kinetic analysis revealed different Km and Vmax values for each rHaGST, suggesting that they have different conjugation rates. The optimum conditions (pH, temperature) and inhibitory assays of each protein demonstrated different optimal ranges. However, HaGSTµ was highly expressed in the ovary and gill, whereas HaGSTρ was highly expressed in the gill and pouch. mRNA expression of HaGSTρ and HaGSTµ was significantly elevated upon lipopolysaccharide, Poly (I:C), and Edwardsiella tarda challenges in liver and in blood cells as well as with Streptococcus iniae challenge in blood cells. From these collective experimental results, we propose that HaGSTρ and HaGSTµ are effective in detoxifying xenobiotic toxic agents, and importantly, their mRNA expression could be stimulated by immunological stress signals in the aquatic environment.

Understanding chemical allergen potency: role of NLRP12 and Blimp-1 in the induction of IL-18 in human keratinocytes.

Keratinocytes (KCs) play a key role in all phases of skin sensitization. We recently identified interleukin-18 (IL-18) production as useful end point for determination of contact sensitization potential of low molecular weight chemicals. The aim of this study was to identify genes involved in skin sensitizer-induced inflammasome activation and to establish their role in IL-18 production. For gene expression analysis, cells were treated for 6 h with p-phenylenediamine (PPD) as reference contact allergen; total RNA was extracted and examined with a commercially available Inflammasome Polymerase Chain Reaction (PCR) array. Among genes induced, NLRP12 (Nod-like receptor P12) was selected for further investigation. NLRP12 promoter region contains Blimp-1 (B-lymphocyte-induced maturation protein-1)/PRDM1 binding site, and from the literature, it is reported that Blimp-1 reduces NLRP12 activity and expression in monocytes/macrophages. Their expression and role in KCs are currently unknown. To confirm NLRP12 expression and to investigate its relationship with Blimp-1, cells were exposed for different times (3, 6 and 24 h) to the extreme sensitizer 2,4-dinitrochlorobenzene (DNCB) and the strong sensitizer PPD. Allergens were able to induce both genes, however, with different kinetic, with DNCB more rapidly upregulating Blimp-1 and inducing IL-18 production, compared to PPD. NLRP12 and Blimp-1 expression appeared to be inversely correlated: Blimp-1 silencing resulted in increased NLRP12 expression and reduced contact allergen-induced IL-18 production. Overall results indicate that contact allergens of different potency differently modulate Blimp-1/NLRP12 expression, with strong allergen more rapidly downregulating NLRP12, thus more rapidly inducing IL-18 production. Data confirm that also in KCs, NLRP12 has an inhibitory effect on inflammasome activation assessed by IL-18 maturation.

Paeoniflorin suppresses IL-6/Stat3 pathway via upregulation of Socs3 in dendritic cells in response to 1-chloro-2,4-dinitrobenze.

Mounting evidence has suggested that inflammation is associated with IL-6/Stat3 pathway in dendritic cells (DCs) and Th17 cells, which are critical for development of allergic contact dermatitis (ACD). Paeoniflorin (PF) has been clinically proved to be effective in the treatment of inflammatory skin diseases such as ACD. We have previously demonstrated the effect of PF on DCs stimulated with 1-chloro-2,4-dinitrobenze (DNCB) and naïve CD4(+)CD45RA(+) T cells for Th17 cell differentiation. However, whether PF down-regulates IL-6/Stat3 in DCs and Th17 cells remains to be explored. In this study, we show clearly that PF markedly decreases IL-6/Stat3 in DCs stimulated with DNCB at both gene and protein levels compared with control DCs in vitro. Meanwhile, PF up-regulates suppressor of cytokine signaling 3 (Socs3). Such decreased expression of IL-6/Stat3 is abolished in DCs that were transfected with Socs3 short interfering RNA (siRNA). When mice CD4(+)CD45 RA(+) T cells were co-cultured with PF-treated DCs stimulated with/without DNCB, the gene expression of the Th17 cell markers such as retinoic acid-related orphan nuclear hormone receptor γt (RORγt), IL-17A, and IL-23R decreased, in accordance with the less secretions of IL-17 and IL-23 in vitro and in vivo. Finally, the suppressed Th17 differentiation induced by PF can be abolished by additional recombinant mouse IL-6. Our results suggest that the anti-inflammatory mechanisms introduced by depletion of Socs3 expression or inactivation of the negative regulator such as Socs3 may represent a promising strategy for the prevention of ACD.

The involvement of the Toll-like receptor signaling and Nrf2-Keap1 pathways in the in vitro regulation of IL-8 and HMOX1 for skin sensitization.

In vitro gene profiling studies have associated the molecular pathways of Nrf2-Keap1 and Toll-like receptor (TLR) signaling with skin sensitization. In this study, the role of these pathways in the regulation of protein biomarkers for skin sensitization was further elucidated using transient gene knock-down of key components of the signaling cascades in HaCaT cells after exposure to dinitrochlorobenzene (DNCB). The effect of targeting these pathways was established through evaluation of heme oxygenase1 (HMOX1) and interleukin (IL)-8 production. These experiments showed that Nrf2 is not involved in regulating HMOX1 after exposure to DNCB, but that activation of TLR signaling moderates the expression of HMOX1. The regulation of IL-8 depended on Nrf2, but also on the Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-ß (TRIF) adaptor protein in TLR signaling. This study provides new insights into the regulation of HMOX1 and IL-8, but the exact regulating mechanisms remain to be further elucidated.

Oral Administration of 4-Hydroxy-3-Methoxycinnamaldehyde Attenuates Atopic Dermatitis by Inhibiting T Cell and Keratinocyte Activation.

Atopic dermatitis (AD) is a skin condition caused by an imbalance of distinct subsets of T helper cells. Previously, we showed that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) inhibits T cell activation but does not induce apoptosis. Here, we examined the mechanism underlying the inhibitory effect of 4H3MC on AD both in vivo and in vitro. We sought to test the pharmacological effects of 4H3MC using a mouse model of 2, 4-'2,4-dinitrocholorobenzene' (DNCB)- and mite-induced AD. Also, we determined whether 4H3MC affects T cell differentiation and proliferation. Oral administration of 4H3MC attenuated the symptoms of DNCB- and mite-induced AD, including increased ear thickness, serum IgE levels, immune cell infiltration into inflammatory lesions, and pathogenic cytokine expression in ear tissues. In vitro, 4H3MC blocked T cell differentiation into Th1 and Th2 subtypes, as reflected by suppression of T-bet and GATA3, which are key transcription factors involved in T cell differentiation. In addition, 4H3MC downregulated T cell proliferation during Th1 and Th2 differentiation and keratinocyte activation. Collectively, these findings suggest that 4H3MC ameliorates AD symptoms by modulating the functions of effector T cells and keratinocytes.

Contact sensitizer 2,4-dinitrochlorobenzene is a highly potent human TRPA1 agonist.

2,4-Dinitrochlorobenzene (DNCB) is widely used in human clinical studies and in experimental animal studies to evoke allergic contact dermatitis. 2,4-Dinitrochlorobenzene is a potent immunogen capable of inducing contact sensitization in all humans exposed. However, the mechanism by which DNCB evokes such symptoms is presently unknown. TRPA1 is a nonselective cation channel that is expressed in peptidergic sensory neurons and fibroblasts. TRPA1 activation was recently implicated in the pathophysiology of atopic dermatitis especially in transducing cutaneous itch signals. Here, we test the hypothesis that DNCB acts as a TRPA1 agonist and thereby evokes allergic symptoms. We found that DNCB activates human TRPA1 dose dependently in FLIPR experiments with an EC50 of 167 nM, an effect that was fully blocked by selective TRPA1 antagonists Chembridge-5861528 and A-967079. Similarly, DNCB activated nonselective TRPA1 current in patch clamp studies. Neutralization of 3 critical cysteines in TRPA1 resulted in a loss of DNCB agonism.

Development of LLNA:DAE: a new local lymph node assay that includes the elicitation phase, discriminates borderline-positive chemicals, and is useful for cross-sensitization testing.

We developed a new local lymph node assay (LLNA) that includes the elicitation phase termed LLNA:DAE for discrimination of borderline-positive chemicals as classified by the LLNA modified by Daicel based on ATP content (LLNA:DA) and for cross-sensitization testing. Although the LLNA:DA method could help identify skin sensitizers, some skin irritants classified as non-sensitizers by the LLNA were classified as borderline positive. In addition, the evaluation for the cross-sensitization potential between chemicals was impossible. In the LLNA:DAE procedure, test group of mice received four applications of chemicals on the dorsum of the right ear for induction and one application on the dorsum of the left ear for elicitation. Control group of mice received one chemical application on the dorsum of the left ear. We evaluated the sensitizing potential by comparing the weights of the lymph nodes from the left ears between the two groups. The results of using the LLNA:DAE method to examine 24 chemicals, which contained borderline-positive chemicals, were consistent with those from the LLNA method, except for nickel chloride (NiCl2). Two chemical pairs, 2,4-dinitrochlorobenzene (DNCB) with 2,4-dinitrofluorobenzene (DNFB) and hydroquinone (HQ) with p-benzoquinone (p-BQ), showed clear cross-sensitization with each other, while another chemical pair, DNFB with hexylcinnamic aldehyde (HCA) did not. Taken together, our results suggest that the LLNA:DAE method is useful for discriminating borderline-positive chemicals and for determining chemical cross-sensitization.

Effect of simultaneous exposure to mixture of two skin sensitizers on skin sensitization response in guinea pigs and mice.

Skin sensitization resulting in allergic contact dermatitis is a common occupational health issue. In this study, the effect of mixing two skin sensitizers on the skin sensitization response was investigated. Skin sensitizers are generally classified into T helper type 1 (Th1) or T helper type 2 (Th2), depending on the induced cytokine profile. Dinitrochlorobenzene (DNCB) and oxazolone (Oxa) are Th1 skin sensitizers and phthalic anhydride (PA) and toluene diisocyanate (TDI) are Th2 skin sensitizers. We investigated the effect on skin sensitization response to mixtures of three pairs of these sensitizers: DNCB and Oxa, DNCB and PA, and PA and TDI, using guinea pig maximization test and mouse ear swelling test. In guinea pigs sensitized with the mixture of DNCB and Oxa or PA and TDI, there were changes of skin sensitization response to DNCB and Oxa, and that to PA. On the other hand, there was no mixture effect in guinea pigs sensitized with the mixture of DNCB and PA. The skin sensitization responses were decreased in mice sensitized with the mixtures of DNCB and Oxa or PA and TDI, whereas the mixture effect was not observed in mice sensitized with the mixture of DNCB and PA. The present findings revealed that mixture effect on the skin sensitization response was observed after simultaneous exposure to two skin sensitizers, and the effect was determined by combinations of mixed skin sensitizers.

Role of PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in THP-1 cells.

We previously demonstrated an age-related decrease in receptor for activated C-kinase (RACK-1) expression and functional deficit in Langerhans cells' responsiveness. This defect specifically involves the translocation of protein kinase C (PKC)-ß. The purpose of this study was to investigate the role of RACK-1 and PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in the human promyelocytic cell line THP-1 and primary human dendritic cells (DC). Dinitrochlorobenzene, p-phenylenediamine and diethyl maleate were used as contact allergens. The selective cell-permeable inhibitor of PKC-ß and the broad PKC inhibitor GF109203X completely prevented chemical allergen- or lipopolysaccharide (LPS)-induced CD86 expression and significantly modulated IL-8 release (50 % reduction). The selective cell-permeable inhibitor of PKC-ε (also known to bind to RACK-1) failed to modulate allergen- or LPS-induced CD86 expression or allergen-induced IL-8 release, while modulating LPS-induced IL-8 release. The use of a RACK-1 pseudosubstrate, which directly activates PKC-ß, resulted in dose-related increase in CD86 expression and IL-8 release. Similar results were obtained with human DC, confirming the relevance of results obtained in THP-1 cells. Overall, our findings demonstrate the role of PKC-ß and RACK-1 in allergen-induced CD86 expression and IL-8 production, supporting a central role of PKC-ß in the initiation of chemical allergen-induced DC activation.

Sophoricoside isolated from Sophora japonica ameliorates contact dermatitis by inhibiting NF-κB signaling in B cells.

Sophoricoside (SOPH) is an isoflavone isolated from Sophora japonica (Leguminosae). In this study, the inhibitory effect of SOPH on contact dermatitis was investigated. At dosages of 3 and 10 mg/kg, SOPH ameliorated 2,4-dinitrochlorobenzene-induced acute and chronic contact dermatitis by 50-70%. As cellular targets, SOPH mainly affected the functions of B cells rather than T cells, macrophages and dendritic cells. As signaling targets, SOPH inhibited the phosphorylation and degradation of IκBα/ß and the nuclear translocation of NF-κB p65 in B cells, but not in dendritic cells and macrophages. SOPH did not affect the phosphorylation of ERK, p38, and JNK MAPKs, in B cells, dendritic cells, and macrophages. Taken together, these results suggest that SOPH ameliorates contact dermatitis by inhibiting mainly NF-κB signaling in B cells.

Attenuated Bordetella pertussis BPZE1 protects against allergic airway inflammation and contact dermatitis in mouse models.

BACKGROUND: We previously reported that prior nasal administration of highly attenuated Bordetella pertussis BPZE1 provides effective and sustained protection against lethal challenge with influenza A viruses. The protective effect was mediated by suppressing the production of major pro-inflammatory mediators. To further explore the anti-inflammatory properties of BPZE1, we investigated the effect of BPZE1 nasal pretreatment on two mouse models of allergic disease, allergic airway inflammation, and contact hypersensitivity (CHS). METHODS: Allergic reactions were induced in mice nasally pretreated with live attenuated BPZE1 bacteria using the ovalbumin (OVA)-induced allergic airway inflammation and dinitrochlorobenzene (DNCB)-induced CHS models. RESULTS: Prior BPZE1 nasal treatment suppressed OVA-induced lung inflammation and inflammatory cell recruitment and significantly reduced IgE levels and cytokine production. Similarly, BPZE1 nasal pretreatment markedly inhibited ear swelling, skin inflammation, and production of pro-inflammatory cytokines in the DNCB-induced CHS model. For both models, we showed that BPZE1 pretreatment does not affect the sensitization phase. Upon challenge, BPZE1 pretreatment selectively reduced the level of cytokines whose production is increased and did not affect the basal level of other cytokines. Together, our observations suggest that BPZE1 pretreatment specifically targets those cytokine-producing effector cells that are recruited and involved in the inflammatory reaction. CONCLUSION: Our study demonstrates the broad anti-inflammatory properties of the attenuated B. pertussis BPZE1 vaccine candidate and supports its development as a promising agent to prevent and/or treat allergic diseases.

A quantitative approach to assess the potency of skin sensitizers in the elicitation phase.

The concept that thresholds exist for the induction of allergic contact dermatitis by chemicals with skin sensitizing properties has been used for a quantitative risk assessment approach. In this approach the potency of skin sensitizers as determined in the Local Lymph Node Assay is used to calculate the threshold for induction of sensitization. These are then used to estimate safe exposure levels for consumers. Whether these exposure levels will protect subjects that are already sensitized is unknown. The elicitation of allergic contact dermatitis supposedly occurs above a certain threshold as well and this threshold is most likely lower than that for the induction. It is unclear if induction thresholds can be extrapolated to elicitation thresholds. The aim of this study was to assess the potency of sensitizers with different sensitizing potencies in the elicitation phase in a mouse model for elicitation. Mice were sensitized by topical application on days 0 and 7 using equipotent concentrations of oxazolone, 2,4-dinitrochlorobenzene (DNCB) and eugenol to ensure that the sensitization strength would not influence the elicitation potency. Mice were challenged on day 21 by topical application on the ears in a dose-dependent manner and dose-response data were used to calculate the elicitation potency. Unexpectedly, sensitizers with different sensitizing potencies induced not the same dose-response curves in sensitized mice. The most potent sensitizer in the elicitation phase was oxazolone, followed by DNCB and eugenol. Similar to the induction phase, under equipotent sensitization conditions strong sensitizers such as oxazolone and DNCB elicit allergic reactions at lower concentrations than weak sensitizers such as eugenol. Our results indicate that elicitation thresholds cannot be readily deduced from sensitization thresholds.

Identification of contact and respiratory sensitizers according to IL-4 receptor α expression and IL-2 production.

Identification of allergenic chemicals is an important occupational safety issue. While several methods exist to identify contact sensitizers, there is currently no validated model to predict the potential of chemicals to act as respiratory sensitizers. Previously, we reported that cytometry analysis of the local immune responses induced in mice dermally exposed to the respiratory sensitizer trimellitic anhydride (TMA 10%) and contact sensitizer dinitrochlorobenzene (DNCB 1%) could identify divergent expression of several immune parameters. The present study confirms, first, that IgE-positive B cells, MHC class II molecules, interleukin (IL)-2, IL-4 and IL-4Rα can differentiate the allergic reactions caused by high doses of strong respiratory (TMA, phthalic anhydride and toluene diisocyanate) and contact sensitizers (DNCB, dinitrofluorobenzene and oxazolone). The second part of the study was designed to test the robustness of these markers when classing the weakly immunogenic chemicals most often encountered. Six respiratory allergens, including TMA (2.5%), five contact allergens, including DNCB (0.25%), and two irritants were compared at doses of equivalent immunogenicity. The results indicated that IL-4Rα and IL-2 can be reliably used to discriminate sensitizers. Respiratory sensitizers induced markedly higher IL-4Rα levels than contact allergens, while irritants had no effect on this parameter. Inversely, contact allergens tended to induce higher percentages of IL-2⁺CD8⁺ cells than respiratory allergens. In contrast, the markers MHC-II, IgE and IL-4 were not able to classify chemicals with low immunogenic potential. In conclusion, IL-4Rα and IL-2 have the potential to be used in classifying a variety of chemical allergens.

Inhibitory effects of Schizandra chinensis extract on atopic dermatitis in NC/Nga mice.

CONTEXT: Schizandra chinensis Baillon (SC) is traditionally used as a medicinal plant in the Orient. Recently, SC has become recognized as an adaptogen by the mainstream medical community. Phytoadaptogens influence respiratory, cardiovascular, uterus myotonic, and immune activities. Atopic dermatitis (AD) is an allergic inflammatory skin disease caused by aberrant and over-reactive immune responses. OBJECTIVE: This study assessed the suppressive effect of SC extract (SCE) on 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD in a NC/Nga mouse model. MATERIALS AND METHODS: AD was induced by topically applying 0.2% DNCB to the hairless-back of NC/Nga mice for 4 weeks. Treated mice received SCE or dexamethasone after AD induction. RESULTS: SCE markedly suppressed DNCB-induced dermatitis, as determined by a count of scratching frequency; measurement of IgE, IgM, and histamine levels in serum; and histological observation of epidermal hyperplasia and mast-cell infiltration. Additionally, SCE lessened DNCB-induced histamine receptor mRNA expression in skin tissue and the splenic expressions of interleukin (IL)-4, IL-5, and high-affinity IgE receptor B protein. CONCLUSION: SCE appears useful for suppression of AD, even though the active pathway(s) remain unknown.

Evaluation of the skin sensitization potential of chemicals in THP-1/keratinocyte co-cultures.

Many attempts have been made to develop in vitro sensitization tests that employ dendritic cells (DCs), DC-like cell lines or keratinocytes. The aim of the present investigation was to establish a co-culture of THP-1 cells and keratinocytes for evaluation of skin sensitization potential of chemicals. Co-cultures were constructed by THP-1 cells cultured in lower compartments and keratinocytes cultured in upper compartments of cell culture inserts. After 24 h exposure to sensitizers (2, 4-dinitrochlorobenzene, p-phenylenediamine, formaldehyde, nickel sulfate, isoeugenol and eugenol) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride and lactic acid), the expression of CD86 and CD54 on THP-1 cells were evaluated by flow cytometry, and cell viabilities were determined. The sensitizers induced the augmentation of CD86 and CD54 expression, but the non-sensitizers had no significant effect. Compared with mono-cultures of THP-1 cells, the augmentation of CD86 and CD54 could be detected even at a non-toxic concentration of sensitizers in THP-1 cell/keratinocyte co-cultures. Moreover, isoeugenol was distinguished as a sensitizer in co-cultures, but failed to be identified in mono-cultures. These results revealed that the co-cultures of THP-1 cells and keratinocytes were successfully established and suitable for identifying sensitizers using CD86 and CD54 expression as identification markers.

Prevention of type 1 diabetes by inducing subclinical dermatitis on a small area.

OBJECTIVE: We have previously epidemiologically shown that type 1 diabetes is inversely associated with contact allergy. This finding is intriguing as type 1 diabetes and contact allergy are two completely different diseases, although T cells are involved in both diseases. The objective of this study was therefore to experimentally study the effect of contact allergens on the development of diabetes in non-obese diabetic mice. METHODS: Non-obese diabetic mice 4 weeks of age were separated into seven groups. One group was exposed to tapped water every 14th day, whereas the remaining six groups were split into sensitizations groups or elicitation groups (exposure every 14th day). These groups were then treated with one of the selected contact allergens (PPD or DNCB) or vehicle (AOO). All groups received the sensitizing treatment regime, and hereafter only the elicitation groups were further treated. If the blood glucose reached 14 mM, the mice were considered diabetic and euthanized. Cardiac heart blood was drawn at euthanization, and a Luminex analysis was done on the serum. RESULTS: We showed that repeated application of a low dose of PPD reduced the incidence of diabetes compared to application with water (47% versus 93%, p=0.004). The rest of the groups developed diabetes with a cumulative incidence rate above 80%. The Luminex cytokine analysis revealed no differences between the groups, and no elevated cytokine level suggested a systemic response. Dermatitis was not noticeable by visual inspection, a histological examination, however, revealed a slight infiltration in the ears in the elicitation groups exposed to contact allergens. CONCLUSION: This study showed that repeated topical application on the ears with a contact allergen could prevent the development of diabetes in non-obese diabetic mice. The contact allergens gave a non-visible, subclinical dermatitis on the application site. Activation of NKT cells to the ear lymph nodes seems to be involved.