Assessment of Lung Eosinophils In Situ Using Immunohistological Staining.

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.

Delivered dose estimate to standardize airway hyperresponsiveness assessment in mice.

Airway hyperresponsiveness often constitutes a primary outcome in respiratory studies in mice. The procedure commonly employs aerosolized challenges, and results are typically reported in terms of bronchoconstrictor concentrations loaded into the nebulizer. Yet, because protocols frequently differ across studies, especially in terms of aerosol generation and delivery, direct study comparisons are difficult. We hypothesized that protocol variations could lead to differences in aerosol delivery efficiency and, consequently, in the dose delivered to the subject, as well as in the response. Thirteen nebulization patterns containing common protocol variations (nebulization time, duty cycle, particle size spectrum, air humidity, and/or ventilation profile) and using increasing concentrations of methacholine and broadband forced oscillations (flexiVent, SCIREQ, Montreal, Qc, Canada) were created, characterized, and studied in anesthetized naïve A/J mice. A delivered dose estimate calculated from nebulizer-, ventilator-, and subject-specific characteristics was introduced and used to account for protocol variations. Results showed that nebulization protocol variations significantly affected the fraction of aerosol reaching the subject site and the delivered dose, as well as methacholine reactivity and sensitivity in mice. From the protocol variants studied, addition of a slow deep ventilation profile during nebulization was identified as a key factor for optimization of the technique. The study also highlighted sensitivity differences within the lung, as well as the possibility that airway responses could be selectively enhanced by adequate control of nebulizer and ventilator settings. Reporting results in terms of delivered doses represents an important standardizing element for assessment of airway hyperresponsiveness in mice.

Assessment of the risk of respiratory sensitization from fragrance allergens released by air fresheners.

Consumers using air fresheners are exposed to the emitted ingredients, including fragrances, via the respiratory tract. Several fragrances are known skin sensitizers, but it is unknown whether inhalation exposure to these chemicals can induce respiratory sensitization. Effects on the immune system were assessed by testing a selection of five fragrance allergens in the respiratory local lymph node assay (LLNA). The probability and extent of exposure were assessed by measuring concentrations of the 24 known fragrance allergens in 109 air fresheners. It was shown that the most frequently used fragrances in air fresheners were D-limonene and linalool. In the respiratory LLNA, these fragrances were negative. Of the other tested chemicals, only isoeugenol induced a statistically significant increase in cell proliferation. Consumer exposure was assessed in more detail for D-limonene, linalool, and isoeugenol by using exposure modeling tools. It was shown that the most frequently used fragrances in air fresheners, D-limonene, and linalool gave rise to a higher consumer exposure compared with isoeugenol. To evaluate whether the consumer exposure to these fragrances is low or high, these levels were compared with measured air concentrations of diisocyanates, known human respiratory sensitizers. This comparison showed that consumer exposure from air fresheners to D-limonene, linalool, and isoeugenol is considerably lower than occupational exposure to diisocyanates. By combing this knowledge on sensitizing potency with the much lower exposure compared to diisocyanates it seems highly unlikely that isoeugenol can induce respiratory sensitization in consumers using air fresheners.

Assessment of the respiratory sensitization potential of proteins using an enhanced mouse intranasal test (MINT).

There remains a need for a simple and predictive animal model to identify potential respiratory sensitizers. The mouse intranasal test (MINT) was developed to assess the relative allergic potential of detergent enzymes, however, the experimental endpoints were limited to evaluation of antibody levels. The present study was designed to evaluate additional endpoints (serum and allergic antibody levels, pulmonary inflammation and airway hyperresponsiveness (AHR)) to determine their value in improving the predictive accuracy of the MINT. BDF1 mice were intranasally instilled on days 1, 3, 10, 17 and 24 with subtilisin, ovalbumin, betalactoglobulin, mouse serum albumin or keyhole limpet hemocyanin; challenged with aerosolized methacholine or the sensitizing protein on day 29 to assess AHR, and sacrificed on day 29 or 30. Under the conditions of this study, evaluation of AHR did not improve the predictive power of this experimental model. Allergic antibody responses and IgG isotype characterization proved to be the most sensitive and reliable indicators of the protein allergenic potential with BAL responses providing additional insight. These data highlight that the evaluation of the respiratory sensitization potential of proteins can be best informed when multiple parameters are evaluated and that further improvements and refinements of the assay are necessary.

Advanced tests for skin and respiratory sensitization assessment.

Sens-it-iv is an FP6 Integrated Project that finished in March 2011 after 66 months of activity, thanks to 12 million € of funding. The ultimate goal of the Sens-it-iv project was the development of a set of in vitro methods for the assessment of the skin and respiratory sensitization potential of chemicals and proteins. The level of development was intended to be at the point to enter the pre-validation phase. At the end of the project it can be concluded that the goal has been largely accomplished. Several advanced methods were evaluated extensively, and for some of them a detailed Standard Operating Procedure (SOP) was established. Other, less advanced methods also contributed to our understanding of the mechanisms driving sensitization. The present contribution, which has been prepared with the support of CAAT-Europe, represents a short summary of what was discussed during the 3-day end congress of the Sens-it-iv project in Brussels. It presents a list of methods that are ready for skin sensitization hazard assessment. Potency evaluation and the possibility of distinguishing skin from respiratory sensitizers are also well advanced.

Decision trees for evaluating skin and respiratory sensitizing potential of chemicals in accordance with European regulations.

Guidance for determining the sensitizing potential of chemicals is available in EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances; REACH guidance from the European Chemicals Agency; and the United Nations Globally Harmonized System (GHS). We created decision trees for evaluating potential skin and respiratory sensitizers. Our approach (1) brings all the regulatory information into one brief document, providing a step-by-step method to evaluate evidence that individual chemicals or mixtures have sensitizing potential; (2) provides an efficient, uniform approach that promotes consistency when evaluations are done by different reviewers; (3) provides a standard way to convey the rationale and information used to classify chemicals. We applied this approach to more than 50 chemicals distributed among 11 evaluators with varying expertise. Evaluators found the decision trees easy to use and recipients (product stewards) of the analyses found that the resulting documentation was consistent across users and met their regulatory needs. Our approach allows for transparency, process management (e.g., documentation, change management, version control), as well as consistency in chemical hazard assessment for REACH, EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances and the GHS.

Considering risks to healthcare workers from glutaraldehyde alternatives in high-level disinfection.

Due to concerns over glutaraldehyde's toxicity, two substitutes have recently been introduced; ortho-phthalaldehyde (OPA), and a mixture of hydrogen peroxide and peracetic acid. There is limited information about the health effects for employees from these products. This study assesses the current practices regarding the use of high-level disinfectants in British Columbian hospitals and predicts the relative toxicities of each product. Industry practices were compiled using a comprehensive survey of current practices and decision processes in all hospitals in British Columbia. Of 95 hospitals, 64 returned surveys; 80% of these used high-level disinfection. Among user hospitals, 49% used glutaraldehyde alone and 51% had introduced alternatives. Concern about staff health was the most common reason for substituting, but this was frequently not considered when choosing specific alternatives. Hospitals that involved occupational health, infection control or regional staff in high-level disinfectant decisions used glutaraldehyde alternatives less often. In most hospitals, it was difficult to find individuals who were knowledgeable about the use of disinfectants. Potential health effects associated with each type of high-level disinfectant were assessed by review of the published literature and available manufacturers' data along with qualitative structure-activity relationship analysis. Results indicated that although all products irritate the skin and respiratory tract, OPA is a potential dermal and respiratory sensitizer but hydrogen peroxide and peracetic acid do not cause allergic reactions. Despite little being known about the risks to employees from glutaraldehyde alternatives, their use is widespread. The potential risks of all high-level disinfectants are serious; thus regulators and users are faced with important risk management decisions before and after they have been introduced into the workplace.

Safety assessment of enzyme-containing personal cleansing products: exposure characterization and development of IgE antibody to enzymes after a 6-month use test.

BACKGROUND: Enzyme-containing personal cleansing products were being considered for the consumer market. Although enzymes have been marketed safely for many years as ingredients in laundry products, their use in a personal cleansing application represented a new type of exposure for consumers that was not supported by the historical safety data. An exposure assessment and additional safety data would be needed before marketing to ensure consumer safety. OBJECTIVE: The work in this paper was designed to evaluate the potential for inhalation exposure to the enzyme during use of this new product while showering. Then a clinical trial was conducted to determine whether or not the level, duration, and routes of exposure encountered during use of this product would induce a Type I sensitization response to the enzyme. METHODS: Exposure was assessed during normal showering activities by collecting air samples with both high volume and personal samplers and quantitating enzyme levels with an ELISA. To assess the potential for sensitization, panelists were asked to use a prototype protease-containing bar product for all personal cleansing tasks and to keep a use diary reporting any associated symptoms. Physical and dermatologic examinations and skin prick tests with enzyme were conducted before the test commenced and at 2-month intervals. RESULTS: Exposure assessment results showed that airborne enzyme levels were primarily dependent on the concentration of the enzyme in the personal cleansing product. Mean values for total airborne enzyme protein ranged from 5.7 to 11.8 ng/m3 when enzyme concentration, time of use, and measurement technique remained constant. After 6 months of at-home product use, four of 61 test subjects using the enzyme-containing bar had positive skin prick test responses when tested with the enzyme. The skin prick test data were supplemented with serologic analyses, which detected IgE specific for the protease enzyme. None of these subjects showed any clinical symptoms indicative of allergic reaction. CONCLUSION: The ability of enzymes to induce development of allergic antibodies in this study led to the conclusion that this prototype enzyme-containing personal cleansing bar would represent an inappropriate use of enzymes in a consumer product application. The likelihood of both induction of an immunologic response and subsequent elicitation of allergy symptoms in a small but significant fraction of the user population was high. This finding resulted in the decision to halt further development of this prototype.

Assessment of respiratory hypersensitivity in guinea pigs sensitized to toluene diisocyanate: improvements on analysis of respiratory response.

Groups of guinea pigs of the Hartley strain were sensitized to toluene diisocyanate (TDI) by combined single intradermal injection and repeated inhalation exposure (3 h/day for 5 consecutive days) to 0, 3.8, 11, 26, 46, and 51 mg TDI/m3 air. One group of animals was sensitized by intradermal injection only. Sham-exposed and TDI-polyisocyanate resin-sensitized guinea pigs served as controls. Three weeks after the first encounter with the inducing agent, animals were challenged with the free TDI (approximately 0.5 mg/m3) and 1 week later with TDI-guinea pig serum albumin conjugate. Breathing patterns were analyzed by objective mathematical procedures taking into account the intensity and duration of the respiratory rate exceeding +/- 3 standard deviations of the individual prechallenge exposure period. In none of the animals challenged with TDI were conclusive immediate-onset respiratory responses identified. During the TDI conjugate challenge a characteristic increase in respiratory rate was observed in all groups sensitized with TDI. In each of the sham and TDI-resin control groups, 1 of 16 animals responded mildly to the conjugate challenge. With regard to analysis of the development of asthma-like dyspnea, the results obtained suggest that respiratory response can suitably be defined by objective mathematical analysis of breathing patterns. Moreover, the "duration" of response exceeding +3 x standard deviation of prechallenge baseline data appears to show less variability when compared to the "intensity" of response (area). It can be concluded that this method of evaluation of respiratory response may be useful to compare more quantitatively this type of data and serves the objective of decreasing potential interlaboratory variability.

A two-centre study for the evaluation and validation of an animal model for the assessment of the potential of small molecular weight chemicals to cause respiratory allergy.

This study evaluated a single intradermal injection model in the guinea pig with subsequent inhalation challenge and serological analysis as a method to predict the potential of chemicals to induce respiratory allergy. Four known respiratory allergens (trimellitic anhydride, diphenyl methane diisocyanate, phthalic anhydride and toluene diisocyanate (TDI)) were screened by two industrial research laboratories using this protocol. Dinitrochlorobenzene, a potent contact allergen, was included as a negative control material. In both laboratories, the respiratory allergens, but not the contact allergen, induced high titre antigen-specific antibodies in treated animals. The inhalation challenge results were similar in both laboratories but were less conclusive in that exposure to free TDI failed to induce pulmonary responses, probably because it fails to penetrate to the deep lung in sufficient concentration. Although the assay shows promise as a means of identifying chemical respiratory sensitisers, its use as a routine screen for the prediction of the ability of materials to induce respiratory allergy in man is probably questionable.

Assessment of respiratory hypersensitivity in guinea-pigs sensitized to diphenylmethane-4,4'-diisocyanate (MDI) and challenged with MDI, acetylcholine or MDI-albumin conjugate.

Guinea-pigs were sensitized to monomeric diphenylmethane-4,4'-diisocyanate (MDI) by two intradermal injections (1-10% MDI, injection volumes of 50-100 microliters/day, on days 0, 2 and 4) or by a single brief high-concentration inhalation exposure (135 or 360 mg/m3, 15 min). Starting with day 21 following sensitization the animals were subjected to inhalation-challenge exposures (30 min) with non-irritating and irritating concentrations of the hapten (MDI). MDI-challenge concentrations ranged from 3.4 +/- 0.9 to 60 +/- 14.3 mg/m3 air. In some groups guinea-pigs were also challenged with acetylcholine (ACh) aerosol or the MDI-guinea pig serum albumin (GPSA) conjugate. Experimental findings indicated that from intradermally sensitized animals an immediate onset respiratory hypersensitivity response could only be elicited with concentrations exceeding the irritant threshold concentration for MDI, i.e. with concentrations greater than approximately 20 mg/m3 air. Guinea-pigs challenged with the MDI-GPSA conjugate (35.3 +/- 2.8 mg/m3 air) also experienced a weak immediate-type respiratory hypersensitivity response. An increased non-specific airway hyper-responsiveness following ACh-challenge was only observed from animals challenged with approximately 60 mg MDI/m3 air. The histopathological evaluation of lungs and lung-associated lymph nodes revealed an association of the increase in eosinophilic granulocytes and concentration of MDI used for challenge exposures. It appeared, in most instances, that this influx was more pronounced in animals sensitized with MDI as compared with concurrent controls challenged with the same MDI concentration. Guinea-pigs sensitized by a single 15-min inhalation exposure to either 135 or 360 mg MDI/m3 air were challenged sequentially with 12 +/- 2.1 mg MDI/m3 air, ACh and MDI-GPSA conjugate. Following the inhalation-induction, an airway hyper-responsiveness was elicited both after challenge with MDI and with the MDI-GPSA conjugate. The influx of eosinophilic granulocytes was more pronounced from animals sensitized by inhalation when compared with guinea-pigs sensitized intradermally and challenged with the same concentration of MDI. Thus, experimental findings suggest that elicitation of respiratory hypersensitivity is concentration-dependent and that challenge concentrations should slightly exceed the threshold concentration for irritation (approximately 20 mg/m3). Sensitization by inhalation increased the susceptibility to irritant stimuli and thus confounds the selection of the most appropriate concentration for challenge. However, the combined assessment of specific pathologic features such as airway eosinophilia and the evaluation of several breathing parameters during hapten- and ACh-challenge make it easier to distinguish effects caused by irritation and respiratory hypersensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Asthma and allergy associated with occupational exposure to ispaghula and senna products in a pharmaceutical work force.

A cross-sectional study of 125 pharmaceutical workers engaged in the manufacture of bulk laxatives based on ispaghula husks (psyllium) and senna pods was conducted. Skin prick tests with extracts of these components revealed that 7.6% were allergic to ispaghula and 15.3% were allergic to senna. Four (3.2%) cases of occupational asthma were identified. The overall prevalence of asthma (6.4%) was less than in a comparable nonexposed Australian population (odds ration, 0.44). Symptoms referrable to the upper airways, eyes, and skin were more prevalent (52.0%) than in the reference population (odds ratio, 1.53). Smokers and nonatopic subjects were more likely to complain of these symptoms if they were sensitized to senna and/or ispaghula than if they were not sensitized (relative risks, 1.9 and 2.6, respectively). Sensitization to ispaghula and/or senna was not a risk factor for asthma. An IgE-mediated allergic mechanism is probably responsible for the allergic symptoms in many of these subjects. Smoking seems to be a cofactor in this process.

[Enzymes are the allergenic components of inhaled pancreatin]. / Enzyme sind die allergenwirksamen Komponenten von inhaliertem Pankreatin.

Pancreatic extracts had caused respiratory disease in five patients. Four of them were not atopic. In four cases alpha-amylase and in two cases trypsin could be identified as causative allergen using RAST and RAST-inhibition. In addition, demonstration of immunologic cross-reactivity between porcine and bovine pancreatin was possible. This most likely can be attributed to the structurally closely related enzymes alpha-amylase and trypsin occurring in both animal species.