Chemical Allergens Research Articles

Atopic Dermatitis in Adults

Atopic dermatitis (AD) is a chronically relapsing eczematous disease, more common in infants and children than in adults and very rare after midlife. The diagnosis of AD is not always easy in adults, especially when the extension of lesions is limited and their distribution atypical. The aim of this study was to investigate epidemiologic and clinical features of adult AD. The medical files of 332 consecutive AD patients were reviewed to ascertain family and personal history of atopy, age at onset, morphology and localization sites of AD lesions, serum total immunoglobulin E levels, skin prick-test and patch-test results. The present study has show that the disease is more frequent in females and during the third decade of life, starts after the 18th year in slightly fewer than half the patients (47.6%), is prevalently localized in the limb flexures, eyelids, and perioral region, but also in the forehead, cheeks, and anterolateral region of the neck, where it is mainly mild to moderate. AD is of the intrinsic type in 30.4% of cases. Positive patch-test reactions to chemical allergens have been observed in 23.8% of patients. These are very important, because they may influence the occupational choices and the development of hand dermatitis.

Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten sensitizers

Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10 μM for 24 and 48 h. The direct-binding haptens dinitrochlorobenzene (DNCB), benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (±S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens.

Chemical allergen induced IgE production and type 2 cytokines expression: Elevated responses in ageing mice

Chemical allergen induced IgE production and type 2 cytokines expression: Elevated responses in ageing mice

Percutaneous penetration characteristics and release kinetics of contact allergens encapsulated in ethosomes

Background: Formulation of the contact allergens dinitrochlorobenzene and isoeugenol in ethanolic liposomes (ethosomes) increases their sensitizing properties in the local lymph node assay compared with an ethanol–water formulation of the allergens. Likewise, isoeugenol and methyldibromo-glutaronitrile formulated in ethosomes enhanced the patch test reactions in sensitized human volunteers. The relationship between the percutaneous penetration/absorption and sensitization/elicitation phases of contact allergy is not well elucidated.Objective: The aim of this study was to investigate whether the observed increased sensitizing and elicitation properties following the formulation of selected contact allergens in ethosomes could be explained by a change in release kinetics of the allergens and their pattern of percutaneous penetration and absorption as well as allergen deposition in epidermis and dermis.Methods: Release kinetics were studied using dialysis bags, and samples were taken at selected time points until equilibrium was reached. Percutaneous absorption and penetration were studied using human skin on Franz cells, and receptor fluid samples were taken at selected time points. Experiments were terminated after 24 hours, and deposition of allergen in epidermis and dermis was measured. Maximum flux and lag time were calculated.Results: Ethosome formulation decreased the release of both allergens compared with the ethanol–water formulation. Ethosome formulation of dinitrochlorobenzene increased its percutaneous penetration but reduced the percutaneous penetration of isoeugenol compared with control formulations. Likewise, all other calculated parameters showed an opposite trend for the 2 allergens in ethosomes and ethanol–water.Conclusions: The present study demonstrates that identical ethosomes affect the percutaneous penetration characteristics of 2 allergens differently. Thus, our results indicate that each combination of an allergen and a vehicle needs to be evaluated separately. The exact mechanistic relationship between percutaneous penetration, release kinetics, and allergenicity of chemicals in various vehicles remains to be clarified.

Prior exposure to organophosphorus and organochlorine pesticides increases the allergic potential of environmental chemical allergens in a local lymph node assay

The dysregulation of immune functions by some pesticides leads to various immune disorders, including immunodeficiency, tumorigenesis, allergies, and autoimmunity. This study's primary objective was to examine the relationship between immune disorders and the immunosuppression induced by immunosuppressive pesticides. We focused on the modulation of allergic potential by the organophosphorus pesticide parathion, organochlorine pesticide methoxychlor, phenoxyacetic acid herbicide 2,4- d-butyl, and benzoic acid fungicide eugenol, as detected by a local lymph node assay (LLNA), which was developed initially for hazard identification of skin sensitization. Parathion and methoxychlor are immunosuppressive chemicals, and 2,4- d-butyl and eugenol are contact allergens. After the immunosuppressive characteristics of parathion and methoxychlor were confirmed in a pilot study, 4-week-old mice were orally administered parathion (0, 0.4, 1.2 mg/kg) or methoxychlor (0, 100, 300 mg/kg). Four weeks after the last administration, an LLNA was conducted using 2,4- d-butyl (0%, 2.5%, 5%, and 10%) and eugenol (0%, 5%, 10%, and 25%). In addition, detailed analysis of their auricular lymph nodes for number of surface antigen expression of T cells and local cytokine production were performed using 5% 2,4- d-butyl and 5% eugenol treatment groups. EC3 values (estimated concentration to yield a stimulation index of 3) of 2,4- d-butyl and eugenol decreased markedly in parathion- and methoxychlor-pretreated groups. Parathion- and methoxychlor-pretreated groups induced marked increase in number of surface antigen expression of T cells and levels of Th1 cytokines (IFN-γ, TNF-α, and IL-17) produced by ex vivo restimulated lymph node cells. According to our results, the allergic potentials of 2,4- d-butyl and eugenol are increased by prior exposure to parathion and methoxychlor.

In vitro and in vivo analysis of pro‐ and anti‐inflammatory effects of weak and strong contact allergens

Inflammation is a crucial step in the development of allergic contact dermatitis. The primary contact with chemical allergens, called sensitization, and the secondary contact, called elicitation, result in an inflammatory response in the skin. The ability of contact allergens to induce allergic contact dermatitis correlates to a great extent with their inflammatory potential. Therefore, the analysis of the sensitizing potential of a putative contact allergen should include the examination of its ability and potency to cause an inflammation. In this study, we examined the inflammatory potential of different weak contact allergens and of the strong sensitizer 2,4,6-trinitrochlorobenzene (TNCB) in vitro and in vivo using the contact hypersensitivity model, the mouse model for allergic contact dermatitis. Cytokine induction was analysed by PCR and ELISA to determine mRNA and protein levels, respectively. Inflammation-dependent recruitment of skin-homing effector T cells was measured in correlation with the other methods. We show that the sensitizing potential of a contact allergen correlates with the strength of the inflammatory response. The different methods used gave similar results. Quantitative cytokine profiling may be used to determine the sensitizing potential of chemicals for hazard identification and risk assessment.

Tracking Human Contact Allergens: From Mass Spectrometric Identification of Peptide-Bound Reactive Small Chemicals to Chemical-Specific Naive Human T-Cell Priming

Modification of proteins by reactive small chemicals is a key step in the activation of chemical-specific T cells in allergic contact dermatitis (ACD). However, an integrated approach to characterize both the precise nature of chemically modified proteins and the chemical-specific T cells is currently lacking. Here, we analyze the molecular conditions for adduct formation of the strong human contact sensitizer 2,4-dinitrochlorobenzene (DNCB) and its water-soluble form, 2,4-dinitrobenzenesulfonic acid (DNBS), with both an all amino acid-containing model peptide (± Cys) and the protein human serum albumin (HSA). Mass spectrometric detection and quantification revealed thiol-dependent peptide adduct formation at all pH values found in human skin layers. Highest modification rates were obtained with DNBS. Accordingly, DNBS- but not DNCB-modified human immature dendritic cells (iDC) induced in vitro primary human T-cell responses as did 2,4,6-trinitrobenzenesulfonic acid-modified iDC as measured by dinitrophenyl (DNP)- and trinitrophenyl (TNP)-specific T-cell proliferation and interferon gamma (IFN-γ) production in CD4(+) and CD8(+) T-cell subsets. Moreover, DNP-modified HSA protein effectively induced primary T-cell responses when processed by iDC. Thus, an integrated approach that combines efficient skin-related in chemico coupling analyses with an in vitro T-cell priming assay can be used to predict in vivo reactions of chemical contact allergens with extracellular and cellular proteins. This strategy supports the development of chemical-specific in vitro assays that are urgently required in predictive hazard identification and risk assessment of allergenic and nonallergenic chemicals.

Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro

The local lymph node assay (LLNA) has been developed to assess skin sensitization, and based on the EC3 value, it can also be used to evaluate allergen potency. Therefore, in the development of in vitro alternatives to the LLNA assay, one should not only consider the hazard identification but also the possibility to classify allergens relatively to their potency. We have recently described a selective release of interleukin-8 (IL-8) by chemical allergens in THP-1 cell line, and identified the activation of p38 mitogen-activated protein kinase (p38 MAPK) as a common pathway. Therefore, the purpose of this study was to expand the number of chemicals tested and to investigate whether IL-8 production and p38 MAPK activation can be used to classify allergens according to their potency. THP-1 cells were exposed to the contact allergens ( p-benzoquinone, 2-aminophenol, isoeugenol, diethyl maleate, citral and imidazolidinyl urea), selected according to their potency in the LLNA, and to lactic acid and propylene glycol as non-sensitizers. p38 MAPK activation was evaluated 5–15 min after treatment by FACS analysis, while IL-8 release was assed by ELISA following 24 h of incubation. p38 MAPK was activated by all contact allergens, including the pro-apten isoeugenol, whereas IL-8 release was significantly increased after stimulation with all allergens tested, except for isoeugenol. The failure of isoeugenol may be due to decrease in the stability of IL-8 mRNA. Irritants exposure, as expected, failed to induce both p38 MAPK activation and IL-8 release. A significant correlation between IL-8 release and the LLNA EC 3 was found (Pearson correlation r = 0.743, p = 0.0036, n = 12). On the contrary, the activation of p38 MAPK showed no significant correlation between LLNA data and vigor of p38 MAPK activation. Overall, data presented confirm our previous observations and reveal IL-8 as potential tool not only to identify sensitizers, with the exception of pro-haptens, but also to classify them according to their potency, while p38 MAPK activation allows the identification of all sensitizers, including pro-haptens, but was not useful for potency classification.

Toxic effects of Senecio brasiliensis on rat phagocytes

s / Toxicology Letters 196S (2010) S37–S351 S199 P205-022 Allergen-stimulated expression of interleukin (IL)-17 isoforms in mice I. Kimber, M. Hayes, R. Dearman University of Manchester, United Kingdom The development of immune and allergic responses is orchestrated by T helper (Th) subsets that are characterized by selective cytokine secretion profiles. Topical exposure of BALB/c strain mice to different classes of chemical allergen has been shown to result in preferential activation of divergent Th cell subsets. Thus, Th1 and Th2 cells are induced by the contact allergen 2,4-dinitrochlorobenzene (DNCB) and the respiratory sensitizer trimellitic anhydride (TMA), respectively. A further subset of inflammatory Th cells that secrete IL-17mayplay an important role in some autoimmune and inflammatory conditions. In the current investigations, the ability of chemical allergens to provoke changes in expression of the expression of IL-17 isoforms has been examined. BALB/c strain mice were exposed to a single topical dose of either 1% DNCB or 25% TMA, or to vehicle alone, for 30min to 72h. At various times thereafter, cytokine production by skin explants (prepared from dorsal halves of ear tissue) or by draining auricular lymph node cells (LNC) was measured by cytokine-specific enzyme-linked immunosorbant assay (ELISA). Topical treatment with DNCB, but not to TMA or vehicle alone, provoked a rapid increase in IL-17A and IL-17F isoforms, peaking at 2–3h. Levels of IL-17F were generally some 5-fold higher than those observed for IL-17A. DNCB-activated LNC also produced high levels of both IL-17A and IL-17F, reaching maximal levels after 6h of exposure. The heterodimer (IL-17A/F) was also detected, but at considerably lower levels. Treatment with TMA induced the same pattern of cytokines in the draining lymph node, but with slightly delayed kinetics, peaking at 48–72h. These data demonstrate that chemical allergens provoke the rapid production of IL-17 isoforms in the skin and draining lymph node which may play a role in the acquisition of sensitization. doi:10.1016/j.toxlet.2010.03.673 P205-023 Toxic effects of Senecio brasiliensis on rat phagocytes F. Elias1, I. Hueza2, A. Latorre1, S. Gorniak1 1 University of Sao Paulo/Research Center of Veterinary Toxicology (CEPTOX), Brazil, 2 Federal University Of Sao Paulo/School of Pharmacy and Biochestry, Brazil Senecio brasiliensis is one of the most toxic poisonous plant species of Senecio’s genus. It contains pyrrolizidine alkaloids (PAs) that are associated with disease in livestock and human beings after be metabolized into the liver in a high reactive electrophiles (pyrrol compound) capable to link with cellular macromolecules, mainly DNA, forming adducts which may initiate acute or chronic toxicity. The development of oxidative stress with the generation of superoxides and lipid-peroxy radicals in PAs toxicity in cattle and cytotoxicity to macrophages mediated by reactive oxygen species were recently documented. Therefore, the aim of this study was to verify if S. brasiliensis induce toxicity in innate immune system cells (macrophages and neutrophils) during phagocytosis activity. PAs was extracted and concentrated into a butanolic residue (BR) and rats were then treated with BR daily, by gavage, during 28 days. Two experiments were performed, one to evaluate peritoneal macrophages and the other to determine peripheral neutrophils activity. For each experiment 40 adult rats were separated into four groups treated with 0.0 (control), 3.0 (A), 6.0 (B), and 9.0 (C)mg/kg of BR. The results obtained showed a significant enhancement in basal (Aand C-group) and PMA induced (C-group) oxidative burst of peritoneal macrophages and also an increase percentage of macrophage phagocytosis (A-, B-, and Cgroup). However, in relation to neutrophils activities, it was only observed an increase in basal oxidative burst (Aand B-group). The intracellular redox potential is maintained in a reduced state by constant elimination of superoxides, peroxides and cellular H2O2 by glutathione (GSH). The pyrrolic metabolites produced from PAs are capable tobindwithGSHto formGSHconjugates. Thus, it is possible to hypothesized that, one of themechanism that S. brasiliensis exerts its immunotoxicity onmacrophage and neutrophil activities is by depleting GSH. doi:10.1016/j.toxlet.2010.03.674 P205-024 Inflammatory response to orthopaedic cobalt chromium alloy wear debris in a rodent air pouch model M. Akbar1, M.H. Grant1, J. Brewer2 1 University of Strathclyde, United Kingdom, 2 University of Glasgow,

Development of a human lung model system for the identification of chemical respiratory allergens in vitro

Development of a human lung model system for the identification of chemical respiratory allergens in vitro

Skin Exposure and Asthma: Is There a Connection?

Numerous occupational and environmental exposures that increase asthma risk have been identified. Research and prevention have focused primarily on the respiratory tract. However, recent studies suggest that the skin may also be an important route of exposure and site of sensitization that contributes to asthma development. Factors that impair skin barrier function, such as filaggrin gene mutations or skin trauma, may facilitate allergen entry and promote Th2-like sensitization and subsequent asthma. Animal studies demonstrate that skin exposure to chemical and protein allergens is highly effective at inducing sensitization, with subsequent inhalation challenge eliciting asthmatic responses. A similar role for human skin exposure to certain sensitizing agents, such as isocyanates, is likely. Skin exposure methodologies are being developed to incorporate skin exposure assessment into epidemiology studies investigating asthma risk factors.

Thioredoxin inhibits epidermal Langerhans’ cell (LC) migration in man and mouse (44.6)

Abstract Epidermal LC play a critical role in the initiation of skin immune responses. LC migration is dependent upon cytokine signals, provided by tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β. We have now examined the ability of human recombinant thioredoxin (hTrx), a redox-active protein, to influence LC migration. Wild type hTrx and various cysteine to alanine mutants were produced in E coli. LC migration in response to chemical contact allergen (oxazolone; Ox) or to intradermal (id) injection of recombinant murine TNF-α or IL-1β was assessed in BALB/c mice. LC migration was provoked in healthy human volunteers (n=10) by id injection of recombinant human TNF-α. Various forms of hTrx were administered by topical application 2h prior to the migratory stimuli. LC densities were assessed in epidermal sheets by immunofluorescent staining for MHC class II (mouse) or CD1a (human). Topical application of hTrx inhibited significantly Ox- and TNF-α-induced LC migration in BALB/c strain mice, but failed to impact on IL-1β-induced migration. Use of mutant forms of hTrx demonstrated that this ability was redox-independent. Application to the skin of wild type hTrx to human volunteers inhibited TNF-α-induced LC migration also. Inhibition of LC migration was selective for IL-1β-dependent stimuli (contact allergen and TNF-α). These data suggest that thioredoxin possesses novel immunomodulatory properties as a result of its ability to modulate IL-1β production and/or signalling.

Allergen Content of Patient Problem and Nonproblem Gloves: Relationship to Allergen-Specific Patch-Test Findings

Identification of putative contact allergen and source material is often done by a combination of patch testing and manufacturer-supplied product information. The accuracy of the identification of allergen-source material and level of allergen in that allergen-source material is not known. The objectives of the study were to survey the chemical allergen content of glove allergic contact dermatitis (ACD) patient-identified problem and nonproblem gloves and to evaluate the ability of the patient to discriminate between problem and nonproblem gloves. Gloves from patch-tested rubber allergen-positive ACD patients were analyzed for species and amount of rubber allergen. Approximately half the subjects were able to correctly identify their problem and nonproblem gloves. Correct association of a glove with ACD was directly related to patch-test reaction severity and inversely related to the number of glove brands being used by the patient. Of note, thiurams were not detected in any of the gloves examined. Although patch testing is invaluable in identifying individual allergen sensitivities, the identification of the ACD-causative specific chemical allergen and source material remains problematic. All glove brands used within days prior to and during an ACD episode should be considered potential sources of the contact allergen.

Development Of In Vitro T Cell Priming Assays For Identification Of Contact Allergens And Respiratory Sensitizers

RATIONALE: In vitro alternatives to replace animal testing in the identification of skin and respiratory sensitizers are urgently needed due to legislation and for ethical reasons. One of the hallmarks of chemical contact allergens and respiratory sensitizers is their ability to induce T cell responses. Our goal is to develop in vitro T cell priming assays within the EU project Sens-it-iv that allow to identify such allergens.

Pre-exposure to sulfur dioxide attenuates most allergic reactions upon trimellitic anhydride challenge in sensitized Brown Norway rats

Irritant-induced inflammation of the airways may aggravate respiratory allergy induced by chemical respiratory allergens. Therefore, it was studied whether airway irritation by sulfur dioxide (SO2) would enhance respiratory allergic reactions to trimellitic anhydride (TMA), using a rat model. Brown Norway (BN) rats were topically sensitized, subsequently exposed for a single time or repeatedly to 300 ppm SO2, and challenged by inhalation to a distinctly irritating or minimally irritating concentration of TMA after the (last) SO2 exposure. Repeated exposure to SO2 alone reduced breathing frequency during exposure, and caused epithelial alterations including hyperplasia and squamous metaplasia, and infiltration of polymorphonuclear inflammatory cells into nasal tissues, larynx, trachea, and bronchi/bronchioli. Histopathological changes were less prominent after 1 day of SO2 exposure. Repeated pre-exposure to SO2 reduced the number of TMA-induced apnoeas, in an SO2 exposure duration-dependent manner. This effect of SO2 on TMA-induced functional allergic reactions (apnoeas) was distinct only when the TMA challenge concentration was not too irritating itself. Repeated pre-exposure to SO2 reduced TMA-induced laryngeal ulceration, goblet-cell hyperplasia, and inflammation in the lungs in most animals, regardless of the TMA challenge concentration. The SO2-induced replacement of normal respiratory epithelium by less sensitive, squamous epithelium may offer an explanation for the, unexpected, reduced allergic manifestation. However in a few animals, SO2 appeared to facilitate TMA-induced irritation, probably due to incomplete protection. Overall, SO2 exposure of TMA-sensitized rats reduced TMA-related allergic respiratory responses in most animals.

IL-6, VEGF, KC and RANTES Are a Major Cause of a High Irritant Dermatitis to Phthalic Anhydride in C57BL/6 Inbred Mice

In previous studies, several strains of mice were used as chemical-induced skin irritation models to identify immunological hazards and elucidate the molecular and cellular mechanisms by which irritant dermatitis disease occur. BALB/c and C57BL/6 mice have been used for most of these experiments. Although there are some differences in the immune response to chemical allergens between these strains, few studies have been conducted to determine what regulatory factors contribute to these variations. To investigate the cause of high responses to skin irritation in C57BL/6 mice that are widely used to study atopic dermatitis, changes in various immune-related factors such as ear thickness, myeloperoxidase activity, lymph node weight, IgE concentration and cytokine concentration were measured in C57BL/6 and BALB/c mice following phthalic anhydride (PA) treatment. Based on analysis of the skin irritation, C57BL/6 mice showed a greater skin irritation to PA than BALB/c mice, although the IgE concentration and auricular lymph node weight did not contribute to this difference in the response. However, the concentration of several cytokines and chemokines (interleukin [IL]-6 and vascular endothelial growth factor [VEGF], keratinocyte-derived chemokine [KC] and regulated on activation normal T cell expressed and secreted [RANTES]) were significantly higher in C57BL/6 mice than BALB/c mice following treatment with PA. Our results suggest that several of the cytokines and chemokines secreted from irritant site could contribute to the regulation mechanism responsible for the difference in the skin irritation among various strains of mice following exposure to PA.

Abrogation of High-Affinity IgE Receptor-Mediated Mast Cell Activation at the Effector Phase Prevents Contact Hypersensitivity to Oxazolone

Inflammatory mediators released from mast cells (MCs) through engagement of the high-affinity receptor for IgE (FcepsilonRI) have pivotal roles in chemical allergen-induced contact hypersensitivity (CHS) reactions, which suggests that the blockade of MC activation through FcepsilonRI stimulation may attenuate allergic contact dermatitis (CD). To address this possibility, we employed the following two approaches: (i) modulation of FcepsilonRI-mediated MC activation by introducing mutations in tyrosine residues of the FcepsilonRI beta-chain immunoreceptor tyrosine-based activation motif (ITAM) and (ii) blockade of FcepsilonRI-mediated MC activation employing a recombinant soluble ecto-domain of the human FcepsilonRIalpha-chain (rsFcepsilonRIalpha). In this study, we show that optimal MC activation through the FcepsilonRI beta-chain ITAM has essential roles in the onset of CHS to oxazolone (Oxa), a well-characterized chemical allergen. In addition, we demonstrate that administration of the rsFcepsilonRIalpha after sensitization successfully prevents murine CHS to Oxa. In a chronic CD model elicited by multiple challenges with low-dose Oxa, application of the rsFcepsilonRIalpha during the course of the challenges showed suppressive effects on CHS to Oxa. Taken together, our data indicate that inhibition of FcepsilonRI-dependent MC activation can suppress allergic CD.

Development of a human lung model system for the identification of chemical respiratory allergens in vitro

Development of a human lung model system for the identification of chemical respiratory allergens in vitro

Use of dendritic cells for the identification and characterization of chemical allergens

Use of dendritic cells for the identification and characterization of chemical allergens

IL-8 release and p38 MAPK activation: Potential tools to classify chemical allergens?

IL-8 release and p38 MAPK activation: Potential tools to classify chemical allergens?