The Safety Assessment of Cosmetic Perfumes by Using In Chemico and In Vitro Methods in Combination with GC-MS/MS Analysis.

Animal testing has been prohibited for the safety assessment of cosmetic ingredients or finished products. Thus, alternative non-animal methods, followed by confirmatory clinical studies on human volunteers, should be used as the sole legally acceptable approach within the EU. The safety assessment of cosmetic products requires the involvement of multiple scientific disciplines, including analytical chemistry and biomedicine, as well as in chemico, in vitro and in silico toxicology. Recent data suggest that fragrance components may exert multiple adverse biological effects, e.g. cytotoxicity, skin sensitisation, (photo)genotoxicity, mutagenicity, reprotoxicity and endocrine disruption. Therefore, a pilot study was conducted with selected samples of fragrance-based products, such as deodorant, eau de toilette and eau de parfum, with the aim of integrating results from a number of alternative non-animal methods suitable for the detection of the following toxicological endpoints: cytotoxicity (with 3T3 Balb/c fibroblasts); skin sensitisation potential (in chemico method, DPRA); skin sensitisation potential (LuSens in vitro method, based on human keratinocytes); genotoxicity potential (in vitro Comet assay with 3T3 Balb/c cells); and endocrine disruption (in vitro YES/YAS assay). The presence of twenty-four specific known allergens in the products was determined by using GC-MS/MS. The strategies for estimation of the NOAEL of a mixture of allergens, which were proposed by the Scientific Committee on Consumer Products in their 'Opinion on Tea tree oil' document and by the Norwegian Food Safety Authority in their 'Risk Profile of Tea tree oil' report, were used as models for the NOAEL estimation of the mixtures of allergens that were identified in the individual samples tested in this study.

Associations between grass pollen exposures in utero and in early life with food allergy in 12-month-old infants.

Birth during pollen seasons may influence food allergy risk but no study has assessed pollen exposure. Using the HealthNuts population-based cohort of 5276 infants, we assessed grass pollen exposures, in utero and up to the first 6 months of life, on hen's egg, sesame and peanut allergy outcomes at 12 months. Cumulative pollen exposure in the first 7 days of life increased risk of peanut sensitization aMOR (adjusted multinomial odds ratio) = 1.21 (95% CI: 1.01-1.44). Exposure between first 4-6 months of life increased risk of hen's egg aMOR = 1.02 (95% CI: 1.004-1.04) and sensitization to all foods aMOR = 1.02 (95% CI: 1.003-1.04). Grass pollen exposure was associated with food challenge diagnosed food allergy, but only among infants with a maternal history of food allergy. Exposure to grass pollen in the intrauterine period and infancy may be important but more studies are needed to replicate these findings.

The association between outdoor allergens - pollen, fungal spore season and high asthma admission days in children and adolescents.

Periods when asthma admissions peaks have serious implications for asthma sufferers and hospitals. We assessed the association between aeroallergen exposure and childhood asthma peak periods during two grass pollen seasons using the Melbourne Air Pollen Children and Adolescent Health (MAPCAH) study conducted in Melbourne, Australia. Two peak periods were identified. Effect modifications by atopy and sex were considered. All pollen 2 days prior was associated with increased odds of these peak periods. Same day fungal spores, but not pollen, were important. Grass at lag 2 was associated with increased odds 1.03 (95%CI 1.01, 1.05) as was the same day Alternaria 1.02 (1.00, 1.04) per spore/m3 for boys. In addition to pollen, fungal spores particularly Alternaria may result in days of high exacerbations during pollen seasons. Further guidance is needed to better prepare families/carers with information about the increased risk of asthma attacks in children prior to pollen seasons.

N-Acetylcysteine improves intestinal function and attenuates intestinal autophagy in piglets challenged with ß-conglycinin.

ß-Conglycinin (ß-CG), an anti-nutritional factor, is a major allergen in soybeans to induce intestinal dysfunction and diarrhea in neonatal animals, including piglets and human infants. This study with a piglet model determined the effects of N-acetylcysteine (NAC) on intestinal function and autophagy in response to ß-CG challenge. Twenty-four 12-day-old piglets (3.44 ± 0.28 kg), which had been weaned at 7 days of age and adapted for 5 days after weaning, were randomly allocated to the control, ß-CG, and ß-CG + NAC groups. Piglets in the control group were fed a liquid diet containing 10% casein, whereas those in the ß-CG and ß-CG + NAC groups were fed the basal liquid diets containing 9.5% casein and 0.5% ß-CG for 2 days. Thereafter, pigs in the ß-CG + NAC group were orally administrated with 50 mg (kg BW)-1 NAC for 3 days, while pigs in the other two groups were orally administrated with the same volume of sterile saline. NAC numerically reduced diarrhea incidence (- 46.2%) and the concentrations of hydrogen peroxide and malondialdehyde, but increased claudin-1 and intestinal fatty-acid binding protein (iFABP) protein abundances and activities of catalase and glutathione peroxidase in the jejunum of ß-CG-challenged piglets. Although ß-CG challenge decreased the villus height, villus height/crypt depth ratio, and mRNA levels of claudin-1 and occludin, no significant differences were observed in these indices between the control and ß-CG + NAC groups, suggesting the positive effects of NAC supplementation on intestinal mucosal barrier function. Moreover, NAC increased the concentrations of citrulline and D-xylose in the plasma, as well as the expression of genes for aquaporin (AQP) 3, AQP4, peptide transporter 1 (PepT1), sodium/glucose co-transporter-1 (SGLT-1), potassium inwardly-rectifying channel, subfamily J, member 13 (KCNJ13), and solute carrier family 1 member 1 (SLC1A1) in the jejunum, demonstrating that NAC augmented intestinal metabolic activity and absorptive function. Remarkably, NAC decreased Atg5 protein abundance and the LC3II/LC3I ratio (an indicator of autophagy) in the jejunum of ß-CG-challenged piglets. Taken together, NAC supplementation improved intestinal function and attenuated intestinal autophagy in ß-CG-challenged piglets.

Liquid Chromatography Tandem Mass Spectrometry Based Label-Free Quantification Method for Assessment of Allergen-Induced Anaphylactoid Reactions.

Mast cells are essential in mediating inflammatory processes. When activated, mast cells can rapidly release characteristic granules and various mediators into the interstitium. Tryptase (TPS) and ß-hexosaminidase (HEXB) are typical protease mediators stored in granules and released upon activation. They have been recognized as important biomarkers of anaphylaxis, and the released level is associated with the severity of allergic reactions. In this study, a sensitive, accurate, and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneously quantifying the two biomarkers was developed and validated in LAD2 cell culture supernatant, and P14R was used as internal standard. Good linearity was observed in the range of 50-2500 ng/mL for TPS and 10-2000 ng/mL for HEXB both with R2 > 0.99. The matrix effect and recovery were both within acceptable limits. We quantified TPS and HEXB released from Laboratory of Allergic Disease 2 (LAD2) mast cells treated with several potential allergens, and the results demonstrate that the method can be used to investigate TPS and HEXB levels in LAD2 mast cell model during allergy research. We anticipate our approach to be a robust and sensitive assessment method for more biomarkers with similar kinetics characteristics and to be a major tool of allergic drug assessment or antiallergic drug development in research.

Sensitivity of KeratinoSensTM and h-CLAT for detecting minute amounts of sensitizers to evaluate botanical extract.

Cosmetic ingredients are often complex mixtures from natural sources such as botanical extracts that might contain minute amounts of constituents with sensitizing potential. The sensitivity of in vitro skin sensitization test methods such as KeratinoSensTM and h-CLAT for the detection of minute amounts of sensitizer in mixtures remains unclear. In this study, we assessed the detection sensitivity of the binary test battery comprising KeratinoSensTM and h-CLAT for minute amounts of sensitizers by comparing the LLNA EC3 (estimated concentration of a substance expected to produce a stimulation index of 3) values to the minimum detection concentrations (MDCs) exceeding the positive criteria for each of the two in vitro test methods. 146 sensitizers with both sets of in vitro data and LLNA data were used. MDC values for KeratinoSensTM and h-CLAT were calculated from exposure concentrations exceeding positive criteria for each in vitro test method (EC1.5 and minimum induction thresholds, respectively). The dilution rate used to expose culture medium was also considered. For 86% of analyzed sensitizers, the in vitro test methods showed MDC values lower than LLNA EC3 values, suggesting that the binary test battery with KeratinoSensTM and h-CLAT have greater sensitivity for detection of minute amounts of sensitizer than LLNA. These results suggest the high applicability of KeratinoSensTM and h-CLAT for detecting skin sensitizing constituents present in botanical extract.

Consensus-based European guidelines for treatment of atopic eczema (atopic dermatitis) in adults and children: part II.

This guideline was developed as a joint interdisciplinary European project, including physicians from all relevant disciplines as well as patients. It is a consensus-based guideline, taking available evidence from other guidelines, systematic reviews and published studies into account. This second part of the guideline covers antimicrobial therapy, systemic treatment, allergen-specific immunotherapy, complementary medicine, psychosomatic counselling and educational interventions, whereas the first part covers methods, patient perspective, general measures and avoidance strategies, basic emollient treatment and bathing, dietary intervention, topical anti-inflammatory therapy, phototherapy and antipruritic therapy. Management of AE must consider the individual clinical variability of the disease. Systemic immunosuppressive treatment with cyclosporine, methotrexate, azathioprine and mycophenolic acid is established option for severe refractory cases, and widely available. Biologicals targeting the T helper 2 pathway such as dupilumab may be a safe and effective, disease-modifying alternative when available. Oral drugs such as JAK inhibitors and histamine 4 receptor antagonists are in development. Microbial colonization and superinfection may cause disease exacerbation and can require additional antimicrobial treatment. Allergen-specific immunotherapy with aeroallergens may be considered in selected cases. Psychosomatic counselling is recommended especially in stress-induced exacerbations. Therapeutic patient education ('Eczema school') is recommended for children and adult patients. General measures, basic emollient treatment, bathing, dietary intervention, topical anti-inflammatory therapy, phototherapy and antipruritic therapy have been addressed in the first part of the guideline.

Ameliorating effects of Nigella sativa oil on aggravation of inflammation, oxidative stress and cytotoxicity induced by smokeless tobacco extract in an allergic asthma model in Wistar rats.

BACKGROUND: The comparison of smokeless tobacco (ST) exposure versus Ovalbumin (Ova) sensitized rats or asthmatic patients has hardly been studied in the literature. Thus, the present study aims to investigate the aggravation of inflammation, exacerbation of asthma, oxidative stress and cytotoxicity induced by ST. METHODS: ST was given at the dose of 40mg/kg in an allergic asthma model in Wistar rats. Furthermore, the effects of oral administration of Nigella sativa oil (NSO), at a dose of 4mL/kg/day, were investigated. RESULTS: The obtained results showed that ST clearly enhanced lung inflammation through interleukin-4 (IL-4) and Nitric oxide (NO) increased production. Actually, ST was found to intensify the oxidative stress state induced by Ova-challenge in rats, which was proven not only by augmenting lipid peroxidation and protein oxidation, but also by altering the non-enzymatic and enzymatic antioxidant status. Furthermore, the aggravation of inflammation and oxidative stress was obviously demonstrated by the histopathological changes observed in lung. In contrast, NSO administration has shown anti-inflammatory effects by reducing IL-4 and NO production, restoring the antioxidant status, reducing lipid peroxidation and improving the histopathological alterations by both protein oxidation and NSO treatment. CONCLUSIONS: Our data have proven that severe concurrent exposure to allergen and ST increases airway inflammation and oxidative stress in previously sensitized rats. They also suggest that the oral NSO treatment could be a promising treatment for asthma.

Preclinical safety and immunological efficacy of Alternaria alternata polymerized extracts.

INTRODUCTION: Alternaria alternata is a widespread fungi whose allergy is a risk factor for asthma development. The use of a polymerized allergen extract (allergoid) may be safer than native extract based treatments while maintaining efficacy. The objective of this study was to characterize biochemically and immunochemically a new Alternaria alternata allergoid. METHODS: Characterization of native and allergoid extracts was performed by determination of protein content, protein and allergenic profile, biological potency, identification of Alternaria allergens, and Alt a 1 quantification. Safety was evaluated in toxicological assays (Ames test, limit test, and fish embryo acute toxicity test in zebrafish, and maximum tolerated dose and Dose-range finding study in rats). Efficacy was evaluated as the capacity to induce IgG antibodies that block IgE-binding to the allergen and cytokine induction (IFN-γ, IL-4, IL-6, IL-10, and TNF-α) in PBMC from atopic donors. RESULTS: Protein and antigenic profiles showed significant modification of the depigmented allergoid with respect to the native extract, inducing a lower IgE binding capacity. Alt a 1, Alt a 3, Alt a 6, and Alt a 8 allergen sequences were identified in the polymer. No toxicological nor genotoxicity effects were observed. The polymer induced IgG antibodies that blocked human IgE binding epitopes, and it induced higher IL-10 levels and similar levels of the other cytokines than native extract in PBMC. CONCLUSIONS: This new A. alternata allergoid could be an effective immunotherapy treatment leading to cytokine stimulation and inducing synthesis of IgG antibodies able to block IgE binding to the allergen. In addition, no toxicological effect was observed, and it may be safer than native extract due to its lower IgE binding capacity and cytokine induction that suggest tolerance induction via T cell shift to Treg (IL-10).

Molecular features of grass allergens and development of biotechnological approaches for allergy prevention.

Allergic diseases are characterized by elevated allergen-specific IgE and excessive inflammatory cell responses. Among the reported plant allergens, grass pollen and grain allergens, derived from agriculturally important members of the Poaceae family such as rice, wheat and barley, are the most dominant and difficult to prevent. Although many allergen homologs have been predicted from species such as wheat and timothy grass, fundamental aspects such as the evolution and function of plant pollen allergens remain largely unclear. With the development of genetic engineering and genomics, more primary sequences, functions and structures of plant allergens have been uncovered, and molecular component-based allergen-specific immunotherapies are being developed. In this review, we aim to provide an update on (i) the distribution and importance of pollen and grain allergens of the Poaceae family, (ii) the origin and evolution, and functional aspects of plant pollen allergens, (iii) developments of allergen-specific immunotherapy for pollen allergy using biotechnology and (iv) development of less allergenic plants using gene engineering techniques. We also discuss future trends in revealing fundamental aspects of grass pollen allergens and possible biotechnological approaches to reduce the amount of pollen allergens in grasses.

Interactive effects of specific fine particulate matter compositions and airborne pollen on frequency of clinic visits for pollinosis in Fukuoka, Japan.

BACKGROUND: Previous studies have revealed the interactive effects of airborne pollen and particulate matter on the daily consultations for pollinosis, but it is uncertain which compositions are responsible. This study aimed to investigate the interactive effects of specific PM2.5 compositions and airborne pollen on the daily number of clinic visits for pollinosis in Fukuoka. METHODS: We obtained daily data on pollen concentrations, PM2.5 compositions, PM2.5 mass, gaseous pollutants (SO2, NO2, CO, and O3), and weather variables monitored in Fukuoka between February and April, 2002-2012. In total, 73,995 clinic visits for pollinosis were made at 10 clinics in Fukuoka Prefecture during the study period. A time-stratified case-crossover design was applied to examine the interactive effects. The concentrations of PM2.5 and its compositions were stratified into low (<15th percentile), moderate (15th-85th percentile), and high (>85th percentile) levels, and the association between airborne pollen and daily clinic visits for pollinosis was analyzed within each level. RESULTS: We found a significant interaction between specific PM2.5 compositions and airborne pollen. Specifically, the odds ratio of daily clinic visits for pollinosis per interquartile increase in pollen concentration (39.8 grains/cm2) at the average cumulative lag of 0 and 2 days during high levels of non-sea-salt Ca2+ was 1.446 (95% CI: 1.323-1.581), compared to 1.075 (95% CI: 1.067-1.083) when only moderate levels were observed. This result remained significant when other air pollutants were incorporated into the model and was fairly persistent even when different percentile cut-off points were used. A similar interaction was found when we stratified the data according to non-sea-salt SO42- levels. This finding differed from estimates made according to PM2.5 and NO3- levels, which predicted that the effects of pollen were strongest in the lower levels. CONCLUSIONS: Associations between airborne pollen and daily clinic visits for pollinosis could be enhanced by high levels of specific PM2.5 compositions, especially non-sea-salt Ca2+.

Theobroma cacao extract attenuates the development of Dermatophagoides farinae-induced atopic dermatitis-like symptoms in NC/Nga mice.

Cacao beans from Theobroma cacao are an abundant source of polyphenols, particularly flavonoids. Previous studies demonstrated that cacao flavanols decrease pro-inflammatory cytokines resulting in the alleviation of allergic symptoms. We sought to investigate the effects of cacao extract (CE) on Dermatophagoides farinae extract (DFE)-induced atopic dermatitis (AD)-like symptoms. CE attenuated DFE-induced AD-like symptoms as assessed by skin lesion analyses, dermatitis score, and skin thickness. Histopathological analysis revealed that CE suppressed DFE-induced immune cell infiltration into the skin. These observations occurred concomitantly with the downregulation of inflammatory markers including serum immunoglobulin (Ig) E, chemokine; thymus and activation-regulated chemokine and macrophage-derived chemokine as well as the skin-derived cytokines interleukin (IL)-4, IL-5, and interferon-γ. CE also significantly alleviated transepidermal water loss and increased skin hydration. These results suggest that CE, a natural phytochemical-rich food, has potential therapeutic efficacy for the treatment of AD.

Safety evaluation of standardized allergen extract of Japanese cedar pollen for sublingual immunotherapy.

Japanese cedar (JC) pollinosis is caused by Japanese cedar pollen (JCP) and most common seasonal allergic disease in Japan. Subcutaneous immunotherapy (SCIT) with allergen extract of JCP (JCP-allergen extract) is well established for JC pollinosis treatment with improvement of symptoms. However, major drawbacks for SCIT are repeated painful injections, frequent hospital visits and anaphylactic risk. Currently, sublingual immunotherapy (SLIT) has received much attention as an advanced alternative application with lower incidence of systemic reactions because the liquid or tablet form of allergen is placed under the tongue. The aim of this study was safety evaluation of standardized JCP-allergen extract currently developed for SLIT in JC pollinosis. JCP-allergen extract showed no potential genotoxicity. No systemic effects were observed in rats administered JCP-allergen extract orally for 26 weeks followed by 4-week recovery period. Mild local reactions such as hyperplasia and increased globule leukocytes resulting from vehicle (glycerin)-induced irritation were observed in stomach. No-observed-adverse-effect level was greater than 10,000 JAU/kg/day for systemic toxicity, equivalent to 300-fold the human dose. No local irritation was found in rabbits oral mucosae by 7-day sublingual administration. These results demonstrate the safe profile of standardized JCP-allergen extract, suggesting it is suitable for SLIT in JC pollinosis.

Utilization of the ex vivo LLNA: BrdU-ELISA to distinguish the sensitizers from irritants in respect of 3 end points-lymphocyte proliferation, ear swelling, and cytokine profiles.

Dermal exposure to chemicals may result in allergic or irritant contact dermatitis. In this study, we performed ex vivo local lymph node assay: bromodeoxyuridine-enzyme-linked immunosorbent assay (LLNA: BrdU-ELISA) to compare the differences between irritation and sensitization potency of some chemicals in terms of the 3 end points: lymphocyte proliferation, cytokine profiles (interleukin 2 [IL-2], interferon-γ (IFN-γ), IL-4, IL-5, IL-1, and tumor necrosis factor α [TNF-α]), and ear swelling. Different concentrations of the following well-known sensitizers and irritant chemicals were applied to mice: dinitrochlorobenzene, eugenol, isoeugenol, sodium lauryl sulfate (SLS), and croton oil. According to the lymph node results; the auricular lymph node weights and lymph node cell counts increased after application of both sensitizers and irritants in high concentrations. On the other hand, according to lymph node cell proliferation results, there was a 3-fold increase in proliferation of lymph node cells (stimulation index) for sensitizer chemicals and SLS in the applied concentrations; however, there was not a 3-fold increase for croton oil and negative control. The SLS gave a false-positive response. Cytokine analysis demonstrated that 4 cytokines including IL-2, IFN-γ, IL-4, and IL-5 were released in lymph node cell cultures, with a clear dose trend for sensitizers whereas only TNF-α was released in response to irritants. Taken together, our results suggest that the ex vivo LLNA: BrdU-ELISA method can be useful for discriminating irritants and allergens.

Exposure to submicron particles (PM1.0) from diesel exhaust and pollen allergens of human lung epithelial cells induces morphological changes of mitochondria tonifilaments and rough endoplasmic reticulum.

In recent literature, little has been said regarding the morphological changes that occur in lung cells after treatment with particles and nanoparticles. Using an in vitro model of type-II lung epithelium (A549), we studied the effects of submicron particles (PM1.0), Parietaria officinalis (ALL), and PM1.0 + ALL together. To date several biochemical effects have been described, instead few data exist in literature regarding morphological events following these treatments, in particular we focused on the morphological changes and distribution of mitochondria, tonifilaments and rough endoplasmic reticulum, using a transmission electron microscopic (TEM) approach. After exposure to PM1.0 particles (PM1.0), Parietaria officinalis as allergen, and PM1.0 with P. officinalis, changes in the cytoplasmic area were observed, such as damage to mitochondria and morphological alterations of the tonifilaments and rough endoplasmic reticulum. The data obtained strongly support the hypothesis that cells in contact with submicron particles (PM1.0), or P. officinalis, undergo alteration of their metabolism.

Oleanolic acid controls allergic and inflammatory responses in experimental allergic conjunctivitis.

Pollen is the most common aeroallergen to cause seasonal conjunctivitis. The result of allergen exposure is a strong Th2-mediated response along with conjunctival mast cell degranulation and eosinophilic infiltration. Oleanolic acid (OA) is natural a triterpene that displays strong anti-inflammatory and immunomodulatory properties being an active anti-allergic molecule on hypersensitivity reaction models. However, its effect on inflammatory ocular disorders including conjunctivitis, has not yet been addressed. Hence, using a Ragweed pollen (RWP)-specific allergic conjunctivitis (EAC) mouse model we study here whether OA could modify responses associated to allergic processes. We found that OA treatment restricted mast cell degranulation and infiltration of eosinophils in conjunctival tissue and decreased allergen-specific Igs levels in EAC mice. Th2-type cytokines, secreted phospholipase A2 type-IIA (sPLA2-IIA), and chemokines levels were also significantly diminished in the conjunctiva and serum of OA-treated EAC mice. Moreover, OA treatment also suppressed RWP-specific T-cell proliferation. In vitro studies, on relevant cells of the allergic process, revealed that OA reduced the proliferative and migratory response, as well as the synthesis of proinflammatory mediators on EoL-1 eosinophils and RBL-2H3 mast cells exposed to allergic and/or crucial inflammatory stimuli such as RWP, sPLA2-IIA or eotaxin. Taken together, these findings demonstrate the beneficial activity of OA in ocular allergic processes and may provide a new intervention strategy and potential therapy for allergic diseases.

Computer models versus reality: how well do in silico models currently predict the sensitization potential of a substance.

National legislations for the assessment of the skin sensitization potential of chemicals are increasingly based on the globally harmonized system (GHS). In this study, experimental data on 55 non-sensitizing and 45 sensitizing chemicals were evaluated according to GHS criteria and used to test the performance of computer (in silico) models for the prediction of skin sensitization. Statistic models (Vega, Case Ultra, TOPKAT), mechanistic models (Toxtree, OECD (Q)SAR toolbox, DEREK) or a hybrid model (TIMES-SS) were evaluated. Between three and nine of the substances evaluated were found in the individual training sets of various models. Mechanism based models performed better than statistical models and gave better predictivities depending on the stringency of the domain definition. Best performance was achieved by TIMES-SS, with a perfect prediction, whereby only 16% of the substances were within its reliability domain. Some models offer modules for potency; however predictions did not correlate well with the GHS sensitization subcategory derived from the experimental data. In conclusion, although mechanistic models can be used to a certain degree under well-defined conditions, at the present, the in silico models are not sufficiently accurate for broad application to predict skin sensitization potentials.

Endothelin-1 concentrations in bronchoalveolar lavage fluid of cats with experimentally induced asthma.

BACKGROUND: There is a need for biomarkers for diagnosis, therapeutic monitoring, and prognosis for asthma in cats. Endothelin-1 (ET-1) is implicated in the pathogenesis of inflammatory airway diseases in other species but not the cat. OBJECTIVE: To conduct a prospective experimental study to show that experimentally asthmatic cats, but not control cats without airway inflammation, would have increased concentrations of ET in BALF. ANIMALS: Eleven healthy, adult research cats. METHODS: Prospective experimental study. Six healthy cats without airway inflammation were used as controls. Asthma was induced using Bermuda grass allergen (BGA) in 5 cats. Collection of BALF for total nucleated cell and differential counts was performed. The concentration of ET-1 in cell-free BALF samples was determined. Data were analyzed using a Mann-Whitney U-test with P < .05 considered significant. RESULTS: The median [range] BALF total cell numbers, eosinophil numbers, and eosinophil percentages were significantly higher in the cats following experimental induction of asthma (1,870 cells/µL [1,450-3,440], 711 cells/µL [356-1,686] and 38% [20-49]) compared to baseline control parameters (462 cells/µL [239-780], 18 cells/µL [18-62] and 3.5% [0-8]) (P < .01). The median [range] BALF ET concentration was also significantly higher after induction of asthma (1.393 fmol/mL[0.977-2.247]) compared to healthy control cats (0.83250 fmol/mL [0.625-1.038]) (P = .012). CONCLUSIONS AND CLINICAL IMPORTANCE: This study suggests that BAL ET-1 concentration can be used to differentiate normal cats from those with experimentally induced asthma. If the same holds true for cats with naturally developing asthma, BAL ET-1 may prove a useful diagnostic biomarker for asthma.

Influence of pollen level on hospitalizations for asthma.

The effect of pollen level on asthma hospitalizations is still under debate. The aim of this study was to analyze hospital admissions due to asthma and its relation with environmental pollen and meteorological factors. During 13 years, we included every patient admitted with asthma as primary or secondary diagnosis. For this purpose, we used a case-crossover analysis to compare pollen concentrations at the time of admission with values of the same variables 2 to 6 days before admission. We included 6,687 hospital admissions. High maximum temperature and low humidity were associated with lower risk of asthma admissions. High mean pollen levels exerted a moderate effect and high maximum pollen levels led to a dramatic increase of hospital admissions due to asthma, especially among females. In conclusion, environmental pollen level increases the risk of asthma hospital admissions.

The sensitivity of the KeratinoSens™ assay to evaluate plant extracts: a pilot study.

Several tests to assess skin sensitization hazard are in peer-review for pre-validation. These tests, as well as the animal tests they aim to replace, were developed (and validated) for the testing of pure substances. However, in the cosmetic field, active ingredients are often mixtures from natural sources. It is therefore important to understand which tests could be used to evaluate their safety. Here we describe a proof-of-concept study to test whether the KeratinoSens(™) assay is able to detect sensitizing constituents within botanical mixtures. Four extracts were spiked with different doses of the sensitizers citral, cinnamic aldehyde and isoeugenol. The tested extracts were negative in the test whereas they became positive in most cases when spiked with the sensitizers. Analysis of the results from the samples spiked with different doses allowed the determination of the minimal level of sensitizers being detectable. The contribution to sensitization potential of doses of 2% and above of the spiked sensitizers were reliably detected. There were limitations for an extract with high cytotoxicity, in which case detection of the artificially spiked sensitizers proved difficult. This study gives a proof of principle for testing of mixtures in the KeratinoSens(™) assay and indicates how sensitive the assay is to detect minor components with sensitizing potential.