IL-32γ suppressed atopic dermatitis through inhibition of miR-205 expression via inactivation of nuclear factor-kappa B.

BACKGROUND: IL-32 is a novel cytokine involved in many inflammatory diseases. However, the role of IL-32γ, an isotype of IL-32, in atopic dermatitis (AD) has not been reported. OBJECTIVE: We investigated the effects of IL-32γ on development of AD and its action mechanisms. METHODS: We used phthalic anhydride (PA) and an MC903-induced AD model using wild-type and IL-32γ transgenic mice. We conducted the therapy experiments by using recombinant IL-32γ protein in a reconstructed human skin model and PA-induced model. We conducted a receiver operating characteristic analysis of IL-32γ with new AD biomarkers, IL-31 and IL-33, in serum from patients with AD. RESULTS: Dermatitis severity and epidermal thickness were significantly reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. The concentration of AD-related cytokines was reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. Subsequent analysis showed that IL-32γ inhibits miR-205 expression in PA- and MC903-induced skin tissue samples and TNF-α/IFN-γ-treated HaCaT cells. IL-32γ reduced NF-κB activity in skin tissue samples from PA- and MC903-induced mice and TNF-α/IFN-γ-treated HaCaT cells. NF-κB inhibitor treatment with IL-32γ expression further suppressed expression of inflammatory mediators as well as miR-205 in TNF-α/IFN-γ-treated HaCaT cells. Furthermore, recombinant IL-32γ protein alleviated AD-like inflammation in in vivo and reconstructed human skin models. Spearman correlation analysis showed that serum levels of IL-32γ and miR-205 were significantly concordant in patients with AD. CONCLUSION: Our results indicate that IL-32γ reduces AD through the inhibition of miR-205 expression via inactivation of NF-κB.

Safety Assessment of Trimellitic Anhydride Copolymers as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 6 trimellitic anhydride copolymers as used in cosmetics. These ingredients are related as copolymers in that they all share trimellitic anhydride (ie, 1,2,4-benzenetricarboxylic acid anhydride) as a monomer, are reported to function as film formers in cosmetics, and are reported to be primarily used in nail products. Very limited safety data were available or submitted. The Panel concluded that Adipic Acid/Neopentyl Glycol/Trimellitic Anhydride Copolymer and Phthalic Anhydride/Trimellitic Anhydride/Glycols Copolymer are safe in nail product formulations in the present practices of use and concentration, but the data are insufficient to make a determination of safety on the use of these 2 ingredients in all other types of cosmetic formulations. The Panel also concluded that the available data are insufficient to make a determination that the remaining trimellitic anhydride copolymers are safe for use in cosmetic formulations.

Anti-inflammatory effect of bee venom in phthalic anhydride-induced atopic dermatitis animal model.

Globally, many people have been affected with atopic dermatitis (AD), a chronic inflammatory skin disease. AD is associated with multiple factors such as genetic, inflammatory, and immune factors. Bee venom (BV) is now widely used for the treatment of several inflammatory diseases. However, its effect on 5% phthalic anhydride (PA)-induced AD has not been reported yet. We investigated the anti-inflammatory and anti-AD effects of BV in a PA-induced animal model of AD. Balb/c mice were treated with topical application of 5% PA to the dorsal skin and ears for induction of AD. After 24 h, BV was applied on the back and ear skin of the mice three times a week for 4 weeks. BV treatment significantly reduced the PA-induced AD clinical score, back and ear epidermal thickness, as well as IgE level and infiltration of immune cells in the skin tissues compared to those of control mice. The levels of inflammatory cytokines in the serum were significantly decreased in BV-treated group compared to PA-treated group. In addition, BV inhibited the expression of iNOS and COX-2 as well as the activation of mitogen-activated protein kinase (MAPK) and NF-Ò¡B induced by PA in the skin tissues. We also found that BV abrogated the lipopolysaccharide or TNF-α/IFN-γ-induced NO production, expression of iNOS and COX-2, as well as MAPK and NF-Ò¡B signaling pathway in RAW 264.7 and HaCaT cells. These results suggest that BV may be a potential therapeutic macromolecule for the treatment of AD.

Piperine Ameliorates Trimellitic Anhydride-Induced Atopic Dermatitis-Like Symptoms by Suppressing Th2-Mediated Immune Responses via Inhibition of STAT6 Phosphorylation.

Atopic dermatitis (AD) is a common inflammatory skin disease predominately related to Type 2 helper T (Th2) immune responses. In this study, we investigated whether piperine is able to improve AD symptoms using a trimellitic anhydride (TMA)-induced AD-like mouse model. Topical treatment with piperine reduced ear swelling (ear thickness and epidermal thickness) induced by TMA exposure. Furthermore, piperine inhibited pro-inflammatory cytokines such as TNF-α and IL-1ß in mouse ears, compared with the TMA-induced AD group. In measuring allergic immune responses in draining lymph nodes (dLNs), we found that IL-4 secretion, GATA3 mRNA level, and STAT6 phosphorylation were suppressed by piperine treatment. In an ex vivo study, piperine also inhibited the phosphorylation of STAT6 on the CD4+ T cells isolated from splenocytes of BALB/c mice, and piperine suppressed IL-4-induced CCL26 mRNA expression and STAT6 phosphorylation in human keratinocytes resulting in the inhibition of infiltration of CCR3+ cells into inflammatory lesions. These results demonstrate that piperine could ameliorate AD symptoms through suppression of Th2-mediated immune responses, including the STAT6/GATA3/IL-4 signaling pathway. Therefore, we suggest that piperine is an excellent candidate as an inhibitor of STAT6 and may help to improve AD symptoms.

Anti-inflammatory effects of Capparis ecuadorica extract in phthalic-anhydride-induced atopic dermatitis of IL-4/Luc/CNS-1 transgenic mice.

CONTEXT: The natural products derived from Capparis ecuadorica H.H. Iltis (Capparaceae) could have great potential for anti-inflammation since they inhibited the inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. OBJECT: This study investigated the anti-inflammatory effects and related mechanism of methanol extract of C. ecuadorica leaves (MCE) during atopic dermatitis (AD) responses. MATERIALS AND METHODS: Alterations in the phenotypical markers for AD, luciferase signal, iNOS-mediated COX-2 induction pathway, and inflammasome activation were analysed in non-Tg (n = 5) and 15% phthalic anhydride (PA) treated IL-4/Luc/CNS-1 transgenic (Tg) HR1 mice (n = 5 per group), subsequent to treatment with acetone-olive oil (AOO), vehicle (DMSO) and two dose MCE (20 and 40 mg/kg) three times a week for 4 weeks. RESULTS: MCE treatment reduced the intracellular ROS level (48.2%), NO concentration (7.1 mmol/L) and inflammatory cytokine expressions (39.1%) in the LPS-stimulated RAW264.7 cells. A significant decrease was detected for ear thickness (16.9%), weight of lymph node (0.7 mg), IgE concentration (1.9 µg/mL), and epidermal thickness (31.8%) of the PA + MCE treated Tg mice. MCE treatment induced the decrease of luciferase signal derived from the IL-4 promoter and the recovery of the IL-4 downstream regulator cytokines. PA + MCE treated Tg mice showed decreasing infiltration of mast cells (42.5%), iNOS-mediated COX-2 induction pathway, MAPK signalling pathway and inflammasome activation in the ear tissue. CONCLUSIONS: These findings provide the first evidence that MCE may have great potential to suppress chemical-induced skin inflammation through the suppression of IL-4 cytokine and the iNOS-mediated COX-2 induction pathway, and activation of inflammasome.

Anti-Inflammatory Effects of Cold Thermal Therapy on Allergic Skin Inflammation Induced by Trimellitic Anhydride in BALB/c Mice.

Cold and hot thermal therapies are widely used as a traditional therapy in many cultures and are often prescribed in the treatment of various musculoskeletal and neurological conditions which present themselves to primary care physicians. However, there are no reports that investigated either the effects of cold and hot thermal therapies on the skin inflammation of trimellitic anhydride- (TMA-) induced dermatitis-like contact hypersensitivity (CHS) mouse model, or the mechanism of thermal therapy on allergic skin inflammation. Therefore, in this study, to reveal the anti-inflammatory effect of thermal therapy and its mechanism on TMA-induced CHS, we analyzed ear-swelling response (ear edema), vascular permeability, serum IgE levels, histological examination, and histamine and Th2 cytokine levels. Cold thermal therapy reduced the ear-swelling response, the vascular permeability, the serum IgE levels, and the infiltration of eosinophils and mast cells as well as the mast cell degranulation. To determine the mechanism by which cold thermal therapy inhibits allergic skin inflammation, detailed studies were carried out revealing that cold thermal therapy suppressed IL-4 and IL-5 secretion and mast cell activation. These results indicated that cold thermal therapy cures skin inflammation of TMA-induced CHS by decreasing Th2 cytokine release, especially IL-4 and IL-5, and mast cell activation. These data suggest that new insight into the mechanism of robust therapeutic effects of cold thermal therapy against allergic dermatitis, and cold thermal therapy may prove to be a useful therapeutic modality on allergic inflammatory diseases as traditional use as well as Th2- or mast cell-mediated allergic responses.

Silkworm dropping extract ameliorate trimellitic anhydride-induced allergic contact dermatitis by regulating Th1/Th2 immune response.

Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by hapten-specific immune response. Silkworm droppings are known to exert beneficial effects during the treatment of inflammatory diseases. Here, we studied whether topical treatment and oral administration of silkworm dropping extract (SDE) ameliorate trimellitic anhydride (TMA)-induced ACD. In ACD mice model, SDE treatment significantly suppressed the increase in both ear thickness and serum IgE levels. Furthermore, IL-1ß and TNF-α levels were reduced by SDE. In allergic responses, SDE treatment significantly attenuated the production of the Th2-associated cytokine IL-4 in both ear tissue and draining lymph nodes. However, it increased the production of the Th1-mediated cytokine IL-12. Thus, these results showed that SDE attenuated TMA-induced ACD symptoms through regulation of Th1/Th2 immune response. Taken together, we suggest that SDE treatment might be a potential agent in the prevention or therapy of Th2-mediated inflammatory skin diseases such as ACD and atopic dermatitis. ABBREVIATIONS: ACD: allergic contact dermatitis; AD: atopic dermatitis; APC: antigen presenting cells; CCL: chemokine (C-C motif) ligand; CCR: C-C chemokine receptor; Dex: dexamethasone; ELISA: enzyme-linked immunosorbent assay; IFN: interferon; Ig: immunoglobulin; IL: interleukin; OVA: ovalbumin; PS: prednisolone; SDE: silkworm dropping extract; Th: T helper; TMA: trimellitic anhydride; TNF: tumor necrosis factor.

Evaluation of chemical-specific IgG antibodies in male workers from a urethane foam factory.

BACKGROUND: Plastic resins are complex chemicals that contain toluene diisocyanate (TDI) and/or trimellitic anhydride (TMA), which cause occupational allergies (OA), including respiratory allergies. Serum IgGs against TDI and TMA have been suggested as potential markers of the exposure status and as exploring cause of OA. Although TDI-specific IgG has been examined for suspected OA, TMA-specific IgG is not commonly evaluated in a urethane foam factory. This study therefore investigated both TDI- and TMA-specific IgGs in suspected OA patients and to evaluate the usefulness of the measurement of multiple chemical-specific IgG measurement for practical monitoring. METHODS: Blood samples were collected from two male workers who developed respiratory allergies supposedly caused by occupational exposure to TDI and/or TMA for the presence of TDI- and TMA-specific IgGs. In addition, blood samples from 75 male workers from a urethane foam factory, along with 87 male control subjects, were collected in 2014 and tested for the same IgGs in 2014. The presence and levels of TDI- and TMA-specific serum IgGs were measured using dot blot assays. RESULTS: We found that controls had mean concentrations of TDI- and TMA-specific IgGs of 0.98 and 2.10 µg/mL, respectively. In the two workers with respiratory allergies, the TDI-specific IgG concentrations were 15.6 and 9.51 µg/mL, and TMA-specific IgG concentrations were 4.56 and 14.4 µg/mL, which are clearly higher than those in controls. Mean concentrations of TDI- and TMA-specific IgGs in the factory workers were 1.89 and 2.41 µg/mL, respectively, and are significantly higher than those of the controls (P < 0.001 and P < 0.026 for TDI- and TMA-specific IgGs, respectively). CONCLUSION: The workers suspected of OA showed an evidently high level of TDI- and TMA-specific IgG, and these levels in workers at the urethane foam factory were also significantly higher than those in controls. In conclusion, the measurement of TDI- and TMA-specific IgG among workers using plastic resins is helpful to monitor their exposure status.

Anti-Inflammatory Effect of Titrated Extract of Centella asiatica in Phthalic Anhydride-Induced Allergic Dermatitis Animal Model.

Centella asiatica has potent antioxidant and anti-inflammatory properties. However, its anti-dermatitic effect has not yet been reported. In this study, we investigated the anti-dermatitic effects of titrated extract of Centella asiatica (TECA) in a phthalic anhydride (PA)-induced atopic dermatitis (AD) animal model as well as in vitro model. An AD-like lesion was induced by the topical application of five percent PA to the dorsal skin or ear of Hos:HR-1 mouse. After AD induction, 100 µL of 0.2% and 0.4% of TECA (40 µg or 80 µg/cm²) was spread on the dorsum of the ear or back skin three times a week for four weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB activity, which were determined by electromobility shift assay (EMSA). We also measured TNF-α, IL-1ß, IL-6, and IgE concentration in the blood of AD mice by enzyme-linked immunosorbent assay (ELISA). TECA treatment attenuated the development of PA-induced atopic dermatitis. Histological analysis showed that TECA inhibited hyperkeratosis, mast cells and infiltration of inflammatory cells. TECA treatment inhibited expression of iNOS and COX-2, and NF-κB activity as well as the release of TNF-α, IL-1ß, IL-6, and IgE. In addition, TECA (1, 2, 5 µg/mL) potently inhibited Lipopolysaccharide (LPS) (1 µg/mL)-induced NO production, expression of iNOS and COX-2, and NF-κB DNA binding activities in RAW264.7 macrophage cells. Our data demonstrated that TECA could be a promising agent for AD by inhibition of NF-κB signaling.

Oral Administration of Achyranthis radix Extract Prevents TMA-induced Allergic Contact Dermatitis by Regulating Th2 Cytokine and Chemokine Production in Vivo.

Allergic contact dermatitis (ACD) remains a major skin disease in many countries, necessitating the discovery of novel and effective anti-ACD agents. In this study, we investigated the preventive effects of Achyranthis radix extract (AcRE) on trimellitic anhydride (TMA)-induced dermatitis and the potential mechanism of action involved. Oral administration of AcRE and prednisolone (PS) significantly suppressed TMA-induced increases in ear and epidermal thickness, and IgE expression. In addition, abnormal expression of IL-1ß and TNF-α protein and mRNA was also significantly attenuated by oral administration of AcRE. Treatment with AcRE also significantly suppressed TMA-induced IL-4 and IL-13 cytokines and mRNA expression in vivo. Moreover, AcRE strongly suppressed TMA-induced IL-4 and IL-5 production in draining lymph nodes, as well as OVA-induced IL-4 and IL-5 expression in primary cultured splenocytes. Interestingly, AcRE suppressed IL-4-induced STAT6 phosphorylation in both primary cultured splenocytes and HaCaT cells, and TMA-induced GATA3 mRNA expression ex vivo. AcRE also suppressed TMA-mediated CCL11 and IL-4-induced CCL26 mRNA expression and infiltration of CCR3 positive cells. The major compounds from AcRE were identified as gentisic acid (0.64 ± 0.2 µg/g dry weight of AcRE), protocatechuic acid (2.69 ± 0.1 µg/g dry weight of AcRE), 4-hydroxybenzoic acid (5.59 ± 0.3 µg/g dry weight of AcRE), caffeic acid (4.21 ± 0.1 µg/g dry weight of AcRE), and ferulic acid (14.78 ± 0.4 ± 0.3 µg/g dry weight of AcRE). Taken together, these results suggest that AcRE has potential for development as an agent to prevent and treat allergic contact dermatitis.

Integrated testing and assessment approaches for skin sensitization: a commentary.

A Bayesian integrated testing strategy (ITS) approach, aiming to assess skin sensitization potency, has been presented, in which data from various types of in vitro assays are integrated and assessed in combination for their ability to predict in vivo skin sensitization data. Here we discuss this approach and compare it to our quantitative mechanistic modeling (QMM) approach based on physical organic chemistry. The main findings of the Bayesian study are consistent with our chemistry-based approach and our previously published assessment of the key determinants of sensitization potency, in particular the relatively high predictive value found for chemical reactivity data and the relatively low predictive value for bioavailability parameters. As it stands at present the Bayesian approach does not utilize the full range of predictive capability that is already available, and aims only to assign potency categories rather than numerical potency values per se. In contrast, for many chemicals the QMM approach can already provide numerical potency predictions. However, the Bayesian approach may have potential for those chemicals where a chemistry modeling approach cannot provide a complete answer (e.g. pro-electrophiles whose in cutaneo activation cannot currently be modeled confidently). Nonetheless, our main message is of the importance of leveraging chemistry insights and read-across approaches to the fullest extent possible.

Trimellitic anhydride facilitates transepithelial permeability disrupting tight junctions in sinonasal epithelial cells.

Trimellitic anhydride (TMA) is a chemical agent classified as a low molecular weight (LMW) agent causing occupational rhinitis (OR) or asthma. Although TMA is recognized as a respiratory sensitizer, the direct and non-immunologic effects of TMA remain unclear. Air- liquid interface (ALI) cultured human nasal epithelial cells (HNECs) derived from control subjects were treated with TMA, followed by measurement of the transepithelial electrical resistance (TEER), paracellular permeability of fluorescein isothiocyanate (FITC)-dextran and immunofluorescence of tight junction proteins claudin-1 and zonula occludens-1 (ZO-1). The cytotoxicity of TMA was evaluated by lactate dehydrogenase (LDH) assay. TMA at concentrations of 2 and 4 mg/mL significantly reduced the TEER within 10 min (p = 0.0177 on 2 mg/mL; p < 0.0001 on 4 mg/mL). The paracellular permeability of FITC-dextran was significantly increased upon challenge with 4 mg/mL TMA for 3 h (p = 0.0088) and 6 h (p = 0.0004). TMA treatment induced a reduction in the fluorescence intensity of claudin-1 and ZO-1 in a dose-dependent manner. LDH assay revealed 4 mg/mL TMA induced cytotoxicity only after 6 h incubation, while 1 or 2 mg/mL TMA caused no cytotoxicity. Our results suggest that TMA has a potential to penetrate the epithelial barrier by disrupting claudin-1 and ZO-1, indicating an important role for sensitization and OR development.

Changes in asthma-like responses after extended removal from exposure to trimellitic anhydride in the Brown Norway rat model.

BACKGROUND: Organic acid anhydride-induced occupational asthma is considered to be IgE-mediated. Airway and skin exposure are the two main routes of sensitization in the work place. Recently we developed an allergic asthmatic Brown Norway rat model sensitized by dermal exposure to trimellitic anhydride (TMA) using an occlusion patch application. OBJECTIVES: The objectives of this study were (1) to develop a model of non-occluded dermal exposure leading to allergic sensitization and (2) to examine the effect of extended removal from exposure on persistence of both specific IgE and TMA aerosol-induced airway responses in this model. METHODS: TMA powder (4 or 40 mg) was applied, unoccluded, to the skin of rats for 4 h, once/week for 4 weeks. Rats were given a 10-min aerosol challenge to 40 mg/m(3) TMA 2 weeks after the last dermal exposure (day 35). Another group was challenged on day 35 and again 18-24 months later. Respiratory enhanced pause (Penh), pulmonary histopathology and inflammation and specific IgE titres were measured. RESULTS: Rats produced dose-dependent specific IgE titres after exposure and developed early-phase (EAR) and late-phase airway responses (LAR) after airway challenge to TMA aerosol as well as airway eosinophilic inflammation. Specific airway responses were still manifested after a second TMA airway challenge given 18-24 months following the initial airway challenge. While persistent, airway inflammation, specific IgE and EAR were significantly attenuated following the second TMA challenge. LAR remained robust at 18-24 months and was not significantly different from the response on day 35. CONCLUSIONS: These results demonstrate the persistence of chemical sensitization and further suggest that IgE is not essential for LAR.

Methylhexahydrophthalic anhydride adducted albumin tryptic peptides in nasal lavage fluid.

Methylhexahydrophthalic anhydride (MHHPA) is a reactive, low molecular weight chemical used in products such as plastics, paints, and electronic components. Exposure to MHHPA may lead to work-related airway diseases such as rhinitis, conjunctivitis, and asthma. Twelve subjects employed at a plant manufacturing electrical capacitors using MHHPA were included in this study. Nasal lavages were collected from subjects before work Monday morning and after work Tuesday afternoon. The levels of MHHPA adducted to serum albumin were analyzed with a straightforward work-up method. The samples were trypsinated before being analyzed with a liquid chromatography-triple quadrupole mass spectrometer. The mass spectrometer was run using selected reaction monitoring for six adducted peptides. Also, some biomarkers of effect (albumin, total protein, eosinophil cationic protein, and tryptase) were analyzed in nasal lavages. Furthermore, the metabolite MHHP acid in urine after work on Tuesday was analyzed by gas chromatography-mass spectrometry. Symptoms from the airways and the eyes and sensitization were registered. The main result of this study is that protein adducts can be analyzed in vivo after low occupational exposures to MHHPA. The results also show a correlation between adducted peptides and albumin in nasal lavage. Furthermore, there may be a difference in the potential to induce hyperresponsiveness between adducts bound to different amino acids.

Exacerbating effects of trimellitic anhydride in ovalbumin-induced asthmatic mice and the gene and protein expressions of TRPA1, TRPV1, TRPV2 in lung tissue.

With the increasing morbidity and mortality of asthma, asthma aggravated by environmental pollution has drawn more attention. This study investigated the exacerbating effects of trimellitic anhydride (TMA), a typical pollutant, in ovalbumin (OVA)-induced asthmatic mice and the gene and protein expressions of TRPA1, V1, V2 in lung tissue. Female BALB/c mice were respectively administered for 42 days as follow: sensitized and challenged with OVA, sensitized and challenged with TMA, sensitized with OVA and challenged with OVA plus TMA, as well as sensitized and challenged with OVA plus TMA. 24 h after the last challenge, the changes in airway resistance (RI) and lung dynamic compliance (Cdyn) were tested. The levels of the inflammatory cells in blood and bronchoalveolar lavage fluid (BALF) were determined. The gene and protein expressions of TRPA1, V1, V2 in lung tissue were examined, and levels of interleukin (IL)-4, -13, substance P (SP), prostaglandin D2 (PGD2), nerve growth factor (NGF) in BALF and the supernatant of lung homogenate were measured. The results indicated that OVA plus TMA significantly increased the amount of inflammatory cells in blood and BALF, enhanced RI while decreased Cdyn, and aggravated lung injury. Increased gene and protein expressions of TRPA1, V1, V2 in lung tissue, level of IL-4 in the supernatant of lung homogenate, levels of IL-13, SP, PGD2, NGF in BALF and the supernatant of lung homogenate were observed. It was suggested that exacerbating effects of TMA in OVA-induced asthma might be related to the regulation of TRPA1, V1, V2 and relevant neurokines.

An in vitro coculture system for the detection of sensitization following aerosol exposure.

The aim of the study was to develop an in vitro model that mimics the alveolar-capillary barrier and that allows assessment of the respiratory sensitizing potential of respiratory sensitizers. The 3D in vitro model cultured at the air liquid interface consists of alveolar type II epithelial cells (A549), endothelial cells (EA.hy926), macrophage-like cells (PMA-differentiated THP-1) and dendritic-like cells (non-differentiated THP-1). This alveolar model was exposed apically to nebulized chemical respiratory sensitizers (Phthalic Anhydride (PA) and TriMellitic Anhydride (TMA)) or irritants (Methyl Salicylate (MeSa) and Acrolein (Acr)) at concentrations inducing at maximum 25% of cytotoxicity. The exposure to respiratory sensitizers induced dendritic cells activation and a specific cytokine release pattern, while the irritants did not. In addition, the cell surface marker OX40L was determined for dendritic like cells activation to identify high molecular weight allergens. With this in vitro model we can postulate a set of promising markers based on the studied compounds that allow the discrimination of chemical respiratory sensitizers from irritants.

Cytokine profiling of chemical allergens in mice: measurement of message versus protein.

Chemical respiratory allergy is an important occupational health problem, but there are currently available no validated methods for hazard identification. There has been interest for some time in the application of cytokine profiling for the characterization of chemical allergens. We have now examined whether these cytokine expression patterns are regulated at the level of mRNA and/or protein production. Mice (BALB/c strain) were exposed topically to the contact allergen 2,4-dinitrochlorobenzene (DNCB), or to the respiratory allergen trimellitic anhydride (TMA). Thirteen days after the initiation of exposure, a single cell suspension of draining (auricular) lymph node cells (LNC) was prepared. Cells were cultured for 24-120h and supernatants analyzed for cytokine protein by cytokine bead array. In parallel experiments total RNA was prepared from freshly isolated or cultured cells and cytokine gene expression was analyzed by ribonuclease protection assay (RPA) or by real time reverse transcription-polymerase chain reaction (RT-PCR). DNCB-activated LNC secreted high levels of the type 1 cytokines interferon (IFN)-gamma and IL-12 compared with TMA-stimulated LNC. The converse type 2 pattern was observed following treatment with TMA. Freshly isolated LNC from TMA-treated mice displayed a selective type 2 cytokine mRNA profile as measured by RPA. In contrast, RNA isolated from DNCB-activated LNC displayed a more mixed cytokine phenotype with relatively low levels of transcripts for both type 1 and type 2 cell products. When cytokine gene expression was measured by the more sensitive real time RT-PCR technique, more vigorous expression of IL-4 was recorded for TMA-activated LNC compared with DNCB-activated LNC but there was no evidence for elevated IFN-gamma transcripts for the latter treatment. The observation that IFN-gamma mRNA expression is not increased in DNCB-activated LNC despite robust secretion of this cytokine indicates that production is controlled mainly at the level of translation of previously transcribed mRNA or of protein secretion. Furthermore, the preferential cytokine profile recorded following TMA exposure was more clearly contrasting when measured at the level of protein secretion rather than at the level of mRNA expression. Experience to date suggests that the measurement of induced cytokine profiles shows promise for the hazard identification and characterization of chemical respiratory allergens, and that the end point best suited for this purpose is cytokine secretion rather than mRNA expression.

Molecular characterization of trimellitic anhydride-induced respiratory allergy in Brown Norway rats.

To contribute to the hazard identification of low molecular weight (LMW) respiratory allergens, respiratory allergy induced by trimellitic anhydride (TMA) was characterized by whole genome analysis of lung tissue and blood proteomics in Brown Norway rats. Dermal sensitization (50% and 25% w/v) with TMA and an inhalation challenge of 15 mg/m(3) TMA-induced apneas, laryngeal inflammation, increased numbers of eosinophils, neutrophils and macrophages in bronchoalveolar lavage (BAL), and increased immunoglobulin E levels in serum and lung tissue. Whole genome analysis of lung, sampled 24 hours after challenge, showed expression changes of not only genes belonging to several Gene Ontology groups with up-regulation of inflammatory-associated genes and those associated with lung remodeling but also genes involved in downsizing these processes. Blood proteomics reflected activation of inflammation-inhibiting pathways. Unsensitized animals challenged with TMA exhibited also an increased number of macrophages in BAL, but gene expression in the above-mentioned gene pathways was unchanged or down-regulated. The authors conclude that parameters for lung remodeling can be a valuable tool in hazard identification of LMW respiratory allergens.

Use of long term dermal sensitization followed by intratracheal challenge method to identify low-dose chemical-induced respiratory allergic responses in mice.

The inhalation of many types of chemicals, including pesticides, perfumes, and other low-molecular weight chemicals, is a leading cause of allergic respiratory diseases. We attempted to develop a new test protocol to detect environmental chemical-related respiratory hypersensitivity at low and weakly immunogenic doses. We used long-term dermal sensitization followed by a low-dose intratracheal challenge to evaluate sensitization by the well-known respiratory sensitizers trimellitic anhydride (TMA) and toluene diisocyanate (TDI) and the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them intratracheally with TMA, TDI, or DNCB, we assayed differential cell counts and chemokine levels in bronchoalveolar lavage fluid (BALF); lymphocyte counts, surface antigen expression of B cells, and local cytokine production in lung-associated lymph nodes (LNs); and antigen-specific IgE levels in serum and BALF. TMA induced marked increases in antigen-specific IgE levels in both serum and BALF, proliferation of eosinophils and chemokines (MCP-1, eotaxin, and MIP-1beta) in BALF, and proliferation of Th2 cytokines (interleukin (IL)-4, IL-10, and IL-13) in restimulated LN cells. TDI induced marked increases in levels of cytokines (IL-4, IL-10, IL-13, and IFN-gamma) produced by restimulated LN cells. In contrast, DNCB treatment yielded, at most, small, nonsignificant increases in all parameters. Our protocol thus detected respiratory allergic responses to low-molecular weight chemicals and may be useful for detecting environmental chemical-related respiratory allergy.

Preexposure to amorphous silica particles attenuates but also enhances allergic reactions in trimellitic anhydride-sensitized brown Norway rats.

Irritant-induced inflammation of the airways may aggravate respiratory allergy induced by chemical respiratory allergens. Therefore, the effect of airway irritation by synthetic amorphous silica (SAS) on respiratory allergy to trimellitic anhydride (TMA) was studied. Brown Norway (BN) rats were topically sensitized on day 0 and on day 7, subsequently exposed for 6 h/day for 6 days to 27 mg/m(3) SAS, and challenged by inhalation to a minimally irritating concentration of 12 mg/m(3) TMA, 24 h after the last SAS exposure. An additional group was exposed to SAS before a second challenge to TMA. Control groups were treated with vehicle, and/or did not receive SAS exposure. Breathing parameters, cellular and biochemical changes in bronchoalveolar lavage (BAL) fluid, and histopathological airway changes 24 h after challenge were the main parameters studied. Exposure to SAS alone resulted in transient changes in breathing parameters during exposure, and in nasal and alveolar inflammation with neutrophils and macrophages. Exposure to SAS before a single TMA challenge resulted in a slightly irregular breathing pattern during TMA challenge. SAS also diminished the effect of TMA on tidal volume, laryngeal ulceration, laryngeal inflammation, and the number of BAL (lung) eosinophils in most animals, but aggravated laryngeal squamous metaplasia and inflammation in a single animal. The pulmonary eosinophilic infiltrate and edema induced by a second TMA challenge was diminished by the preceding SAS exposure, but the number of lymphocytes in BAL was increased. Thus, a respiratory particulate irritant like SAS can reduce as well as aggravate certain aspects of TMA-induced respiratory allergy.