Lisa B. Copeland

A comparison of the allergic responses induced by Penicillium chrysogenum and house dust mite extracts in a mouse model

UNLABELLED A report by the Institute of Medicine suggested that more research is needed to better understand mold effects on allergic disease, particularly asthma development. We compared the ability of the fungal Penicillium chrysogenum (PCE) and house dust mite (HDM) extracts to induce allergic responses in BALB/c mice. The extracts were administered by intratracheal aspiration (IA) at several doses (0, 2.5, 5, 10, 20, 40, and 80 μg) four times over a 4-week period. Three days after the last IA exposure, serum and bronchoalveolar lavage fluid (BALF) were collected. The relative allergenicity of the extracts was evaluated based on the lowest dose able to induce a significant response compared to control (0 μg) and the robustness of the response. PCE induced the most robust response at the lowest dose for most endpoints examined: BALF total, macrophage, neutrophil, and eosinophil cell counts, and antigen-specific IgE. Taken together, our data suggest that PCE may induce a more robust allergic and inflammatory response at lower doses than HDM. PRACTICAL IMPLICATIONS Our data suggest that Penicillium chrysogenum is a robust allergen and may be a more potent allergen source than house dust mite (HDM) in this mouse model. Two critical factors in the development of human allergic disease, exposure levels and sensitization thresholds, are unknown for most allergens including molds/fungi. Human exposure levels are not within the scope of this article. However, the data presented suggest a threshold dose for the induction of allergic responsiveness to P. chrysogenum. Additionally, P. chrysogenum as well as other molds may play an important role in asthma development in our society.

Differential allergy responses to Metarhizium anisopliae fungal component extracts in BALB/c mice

Intratracheal aspiration (IA) exposure to Metarhizium anisopliae crude antigen (MACA), which is composed of equal protein amounts of mycelium (MYC), conidia (CON) and inducible proteases/chitinases (IND) extracts/filtrates, has resulted in responses characteristic of human allergic asthma in mice. The study objective was to evaluate the potential of each component extract to induce allergic/asthma-like responses observed in this mouse model. BALB/c mice received 4 IA exposures to MACA, CON, MYC, IND, or bovine serum albumin (BSA; negative control) or appropriate vehicle control or inflammatory control over a 4-wk period. Mice were assessed by whole-body plethysmography for immediate airway responses and airway hyperresponsiveness to methacholine (Mch) challenge (PenH). Serum and bronchoalveolar lavage fluid (BALF) were collected 3 d after the final exposure. Additionally, BALF neurotrophin levels and extract protease and chitinase activity levels were evaluated. Western blot analysis showed that each component contained different IgE-reactive proteins. All fungal extract exposures resulted in elevated BALF total and differential cell counts, IgE and IgA and total serum IgE compared to HBSS and BSA controls. MYC-exposed mice had the highest responses except for neutrophil influx, which was highest in MACA and IND exposures. However, the MYC-exposed mice had significantly lower PenH values compared to other treatments. By comparison IND and MACA induced significantly higher PenH values. Additionally, IND had substantially higher protease activity levels but induced the lowest neurotrophin levels compared to the other fungal exposures. In this allergic asthma model extract chitinase activity was not associated with allergic responses. In summary, multiple exposures to any of the M. anisopliae component extracts induced allergic/asthma-like responses in BALB/c mice but the response magnitude was different for each component and each appears to contain unique IgE-reactive proteins. Therefore, hazard identification and/or risk assessment for molds must test both mycelia and conidia.

Assessing the allergenic potential of molds found in water-damaged homes in a mouse model

Abstract Damp/moldy indoor environments, which have resulted from flooding events and may increase as a result of climate change, have been associated with asthma exacerbation. Certain molds found in significantly higher or lower concentrations in asthmatics’ homes compared to control homes have been categorized as Group 1 (G1) and Group 2 (G2) molds, respectively. We have compared the allergic potential of selected G1/G2 molds to house dust mite (HDM) in a mouse model. BALB/c mice were exposed to mold (0–80 µg) or HDM (20 µg) extract by intratracheal aspiration either 4X over 4 weeks (allergenicity) or 1X (non-specific responses). Airflow limitation (methacholine challenge) was measured (Day 1) and serum and bronchoalveolar lavage fluid were collected (Day 2) after the final exposure. The G1 molds induced low-to-moderate responses and required higher doses to achieve antigen-specific IgE results similar to those induced by HDM. Compared to HDM responses, the G2 mold in this study required lower doses to induce a similar response. Acute exposure responses suggest some molds may exacerbate asthmatic responses. These studies demonstrate the differing capacities of molds to induce responses associated with allergic asthma, including differences in the threshold dose for allergy induction. Therefore, molds must be evaluated individually for allergic/asthmatic potential. These studies along with our previous studies with G1 (Stachybotrys chartarum)/G2 (Penicillium chrysogenum) molds suggest that the G1/G2 categorization is not indicative of allergic potential but they do not preclude this categorization’s utility in determining unhealthy building dampness.

Biomarkers of acute respiratory allergen exposure: Screening for sensitization potential

Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens following an acute exposure in naïve individuals. Female BALB/c mice received a single intratracheal aspiration exposure to Metarhizium anisopliae crude antigen (MACA) or bovine serum albumin (BSA) in Hanks Balanced Salt Solution (HBSS) or HBSS alone. Mice were terminated after 1, 3, 6, 12, 18 and 24 h. Bronchoalveolar lavage fluid (BALF) was evaluated to determine total and differential cellularity, total protein concentration and LDH activity. RNA was isolated from lung tissue for microarray analysis and qRT-PCR. MACA administration induced a rapid increase in BALF neutrophils, lymphocytes, eosinophils and total protein compared to BSA or HBSS. Microarray analysis demonstrated differential expression of genes involved in cytokine production, signaling, inflammatory cell recruitment, adhesion and activation in 3 and 12 h MACA-treated samples compared to BSA or HBSS. Further analyses allowed identification of approximately 100 candidate biomarker genes. Eleven genes were selected for further assessment by qRT-PCR. Of these, 6 demonstrated persistently increased expression (Ccl17, Ccl22, Ccl7, Cxcl10, Cxcl2, Saa1), while C3ar1 increased from 6-24 h. In conclusion, a single respiratory exposure of mice to an allergenic mold extract induces an inflammatory response which is distinct in phenotype and gene transcription from the response to a control protein. Further validation of these biomarkers with additional allergens and irritants is needed. These biomarkers may facilitate improvements in screening methods.

Maternal respiratory sensitization and gestational allergen exposure does not affect subsequent pup responses to homologous or heterologous allergen

Evidence suggests that the predisposition towards atopy begins early in life. Maternal allergy has been associated with an increased risk of the development of allergic disease in offspring. Some studies suggest that the development of childhood atopy may also be influenced by prenatal allergen exposure. In this study, a respiratory allergen exposure model was used to determine the impact of maternal sensitization (with or without additional exposures during pregnancy) on subsequent pup responses to homologous or heterologous allergen. Female BALB/c mice received two intratracheal aspiration (IA) exposures to Metarhizium anisopliae crude antigen (MACA) or Hank’s buffered salt solution (HBSS) prior to breeding. Some mice also received three additional exposures during pregnancy. Control mothers did not receive treatment. Young adult offspring received three IA exposures to MACA, house dust mite extract (HDM) or HBSS. Offspring sensitized as young adults to either HDM or MACA developed an airway inflammatory response, including increased bronchoalveolar lavage fluid lactate dehydrogenase activity, total protein and total and differential cell counts compared to offspring exposed to HBSS. Increased airway responsiveness to methacholine was observed in pups treated with HDM but not with MACA. Maternal sensitization status (with or without gestational allergen exposure) had no effect on offspring response to either MACA or HDM. In conclusion, this study demonstrates that IA administration of MACA or HDM extract to young adult BALB/c mice induces the development of an inflammatory airway response. In contrast to previous reports, neither maternal sensitization nor gestational allergen exposure could be demonstrated to have a clear effect on offspring sensitization. This discrepancy may be a function of the respiratory sensitization and exposure protocol used in this study, which mimics natural sensitization more closely than do parenteral routes of exposure.

The effects of pregnancy on the exacerbation and development of maternal allergic respiratory disease.

The T-helper 2 (TH2) bias associated with pregnancy may predispose the pregnant mother to the development or exacerbation of allergic disease. To determine the effects of pregnancy on pre-existing maternal sensitization, we sensitized BALB/c mice before breeding by two intratracheal aspiration (IA) exposures to the fungal allergen, Metarhizium anisopliae crude antigen (MACA). Some mice also received three IA exposures to MACA on gestational days 11, 15, and 19. After weaning, all mice were challenged IA with MACA before killing. To determine the effects of pregnancy on susceptibility to future sensitization, naïve parous and nulliparous BALB/c mice were sensitized by three IA exposures to MACA or to Hank’s buffered salt solution vehicle control. Pregnancy did not have a significant effect on individual inflammatory parameters (airway responsiveness to methacholine, total serum and bronchoalveolar lavage fluid (BALF) IgE, BALF total protein, lactate dehydrogenase activity, and total and differential cell counts) following allergen challenge in sensitized mice, regardless of post-breeding allergen exposure. In conclusion there was a weak inhibition of the overall response in mice exposed to allergen during pregnancy compared to identically treated nulliparous mice. In contrast, parous mice that did not encounter allergen post-breeding tended to have exacerbated responses. Parity had no significant impact on future susceptibility to sensitization.

Evaluating Antigen-Specific IgE Using the Rat Basophil Leukemia Cell (RBL) Assay

Allergic diseases (atopy) include asthma, allergic rhinitis, conjunctivitis, and allergic sinusitis. It is estimated that up to 90% of asthmatics are atopic and have an allergy trigger for asthmatic episodes. In order to assess the risk of allergy induction associated with inhalation exposure, animal models of protein allergy have been developed. These models have been used both to identify proteins as allergens and to assess their relative potency. Often these research situations include allergens that are not well characterized or are unknown. In these situations, specific allergens are not available to be evaluated by more well-known assays (such as ELISAs), and developing a specific assay to evaluate an extract or mixture for an unknown or potential allergen is very time consuming and generally requires purified antigen/allergen. Additionally, when the comparison of the relative potency of multiple extracts is of interest, a common/generic platform is necessary. A more generic method, the rat basophil leukemia cell assay (RBL assay), has been developed which provides insight into the allergenicity of extracts and mixtures as well as providing a common platform for relative potency comparison between/among these complex allergen sources.

Differential exposure and acute health impacts of inhaled solid-fuel emissions from rudimentary and advanced cookstoves in female CD-1 mice

Background: There is an urgent need to provide access to cleaner end user energy technologies for the nearly 40% of the worlds population who currently depend on rudimentary cooking and heating systems. Advanced cookstoves (CS) are designed to cut emissions and solid‐fuel consumption, thus reducing adverse human health and environmental impacts. Study premise: We hypothesized that, compared to a traditional (Tier 0) three‐stone (3‐S) fire, acute inhalation of solid‐fuel emissions from advanced natural‐draft (ND; Tier 2) or forced‐draft (FD; Tier 3) stoves would reduce exposure biomarkers and lessen pulmonary and innate immune system health effects in exposed mice. Results: Across two simulated cooking cycles (duration ˜ 3 h), emitted particulate mass concentrations were reduced 80% and 62% by FD and ND stoves, respectively, compared to the 3‐S fire; with corresponding decreases in particles visible within murine alveolar macrophages. Emitted carbon monoxide was reduced ˜ 90% and ˜ 60%, respectively. Only 3‐S‐fire‐exposed mice had increased carboxyhemoglobin levels. Emitted volatile organic compounds were FD ≪ 3‐S‐fire ≤ ND stove; increased expression of genes involved in xenobiotic metabolism (COX‐2, NQO1, CYP1a1) was detected only in ND‐ and 3‐S‐fire‐exposed mice. Diminished macrophage phagocytosis was observed in the ND group. Lung glutathione was significantly depleted across all CS groups, however the FD group had the most severe, ongoing oxidative stress. Conclusions: These results are consistent with reports associating exposure to solid fuel stove emissions with modulation of the innate immune system and increased susceptibility to infection. Lower respiratory infections continue to be a leading cause of death in low‐income economies. Notably, 3‐S‐fire‐exposed mice were the only group to develop acute lung injury, possibly because they inhaled the highest concentrations of hazardous air toxicants (e.g., 1,3‐butadiene, toluene, benzene, acrolein) in association with the greatest number of particles, and particles with the highest % organic carbon. However, no Tier 0–3 ranked CS group was without some untoward health effect indicating that access to still cleaner, ideally renewable, energy technologies for cooking and heating is warranted. HIGHLIGHTSMice inhaled emissions from advanced cookstoves (CS) and a 3‐stone (3‐S)‐fire.Tier 2 and 3 CS emitted significantly less particulate matter and carbon monoxide.3‐S fire (Tier 0)‐exposed mice were the only group to develop acute lung injury.Diminished macrophage phagocytosis was observed in Tier 2 CS‐exposed mice.Lung glutathione was significantly depleted across all Tier 0–3 CS‐exposed mice.