Zana L. Lummus

Quantitative PCR analysis of house dust can reveal abnormal mold conditions

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

Diisocyanate antigen–enhanced production of monocyte chemoattractant protein-1, IL-8, and tumor necrosis factor-α by peripheral mononuclear cells of workers with occupational asthma

BACKGROUND Previous studies have shown a significant association between confirmed diisocyanate-induced asthma (DOA) and in vitro production of diisocyanate antigen-stimulated histamine-releasing factors by PBMCs. Chemokines found in PBMC supernatants are known to express histamine-releasing factor activity. OBJECTIVE PBMCs of diisocyanate-exposed workers were tested in vitro for diisocyanate antigen-specific enhancement of monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-3 (MCP-3), macrophage inflammatory protein-1alpha, RANTES, IL-8, and T-cell cytokines that could play a regulatory role in chemokine synthesis (IL-4, IL-5, IFN-gamma, and TNF-alpha. METHODS Secretion of chemokines and cytokines was determined by quantitative immunochemical assays of PBMC supernatants. Synthesis of mRNA for beta-chemokines was determined by reverse transcription-polymerase chain reaction. RESULTS PBMCs of workers with DOA showed significantly enhanced secretion for MCP-1 compared with diisocyanate-exposed asymptomatic workers (P < .05). In vitro induction of antigen-stimulated MCP-1 mRNA synthesis in cultured PBMCs was demonstrated by reverse-transcription polymerase chain reaction. Quantitation of cytokines in supernatants showed increased mean production of IL-8 and TNF-alpha. IFN-gamma, IL-4, and IL-5 were not enhanced in subjects with DOA. CONCLUSION Antigen stimulation of MCP-1 and TNF-alpha suggest that diisocyanate-specific cellular immune reactions result in activation of macrophages, which may be important in the pathogenesis of DOA.

T-cell receptor Vβ gene segment expression in diisocyanate-induced occupational asthma

BACKGROUND Diisocyanates are the most common cause of occupational asthma induced by low-molecular-weight chemicals. The disease appears to be immunologically mediated but is independent of IgE antibody synthesis. An underlying genetic susceptibility is suggested by the fact that the disease only develops in approximately 5% to 10% of exposed workers. OBJECTIVE The study was designed to determine whether disease susceptibility is influenced by HLA and T-cell receptor V beta gene segment usage. METHODS T-cell receptor V beta repertoires were quantitated by using primer pairs specific for V beta gene segments in conjunction with a common C beta region primer. One group of workers with diisocyanate-induced occupational asthma produced diisocyanate-specific IgG and IgE antibodies, whereas the other group did not produce specific antibodies. Occupational asthma was previously confirmed by either workplace challenge or laboratory specific diisocyanate bronchoprovocation. Control groups consisted of diisocyanate-exposed workers who were free of symptoms, patients with nonoccupational asthma, and unexposed subjects who were free of symptoms. RESULTS Lymphocytes from workers with diisocyanate-induced occupational asthma had significantly decreased V beta 1 and V beta 5 gene segment expression before in vitro exposure to diisocyanates, compared with control groups. Percent V beta 1 and V beta 5 gene segment expression was selectively increased when peripheral blood mononuclear cells were stimulated in vitro with diisocyanate-conjugated proteins. Low-resolution HLA class II phenotyping revealed no significant differences in the distribution of HLA-DR or HLA-DQ alleles between diisocyanate-induced occupational asthma and control groups. CONCLUSIONS These findings are consistent with a hypothesis that antigen-specific T-cell subpopulations may be sequestered in the lungs of workers with diisocyanate-induced occupational asthma and clonally expand after further exposure to diisocyanates.

Diisocyanate conjugate and immunoassay characteristics influence detection of specific antibodies in HDI-exposed workers.

Background The structural characteristics of diisocyanate chemical protein antigens vary depending upon the methods of production, and may influence diisocyanate antigen immunoassays. The impact of different antigen preparation methods on immunoassay sensitivity, specificity, and predictive value for identifying workers with diisocyanate asthma (DA) has not been systematically evaluated.

A cross-sectional survey of sensitization to Aspergillus oryzae–derived lactase in pharmaceutical workers☆☆☆

BACKGROUND The presence of IgE-mediated occupational respiratory sensitization to microbial enzymes has been well documented in a variety of industries. Aspergillus oryzae -derived lactase is used as a dietary aid for patients with lactose intolerance. OBJECTIVE In 1993, a cross-sectional survey of 94 pharmaceutical workers exposed to lactase for a mean duration of 23 months and 24 nonexposed recently hired employees was initiated to identify lactase-sensitized workers and potential risk factors that could be used in making recommendations for preventing future cases of lactase sensitization. METHODS The survey included a physician-administered questionnaire, skin prick testing to lactase enzyme and a panel of common aeroallergens, and spirometry. RESULTS Twenty-seven of 94 lactase-exposed workers (29%) had positive skin test responses to lactase. These workers were 9 times more likely to have upper or lower respiratory symptoms compared with workers with negative skin test responses. Atopic workers were 4 times more likely to have lactase skin sensitivity than nonatopic workers. However, atopy was not a risk factor for the development of upper and/or lower respiratory symptoms. Lactase skin reactivity was not observed in the 24 nonexposed employees. CONCLUSION This cross-sectional survey revealed that atopic workers were more likely to have lactase sensitization and that lactase-sensitized workers were more likely to have upper and/or lower respiratory symptoms, but atopy was not a risk factor for upper or lower respiratory symptoms. In spite of these findings, the company allowed only nonatopic, nonlactase-sensitized workers to continue working in high lactase-exposure areas with careful symptom monitoring and use of protective clothing. Although this strategy was successful in total prevention of new cases of occupational respiratory disease after 5 years, the results of this cross-sectional survey do not support exclusion of atopic workers from working with industrial enzymes.

Interferon-γ Promoter Is Hypermethylated in Blood DNA from Workers with Confirmed Diisocyanate Asthma

Risk factors have not been identified that determine susceptibility for development of diisocyanate-induced occupational asthma (DA). We hypothesized that diisocyanate (DI) exposure could modify gene promoter regions regulating transcription of cytokine mediators and thereby influence expression of DA. A cross-sectional study was designed to investigate the promoter methylation status of candidate genes in DI-exposed workers. Subjects consisted of 131 workers in three groups: 40 cases with DA confirmed by a positive specific inhalation challenge (SIC) (DA+), 41 exposed workers with lower respiratory symptoms and negative SIC (DA-), and 50 asymptomatic exposed workers (AWs). We studied four candidate genes (GSTM1, DUSP22, IFN-γ, and IL-4) for which altered promoter methylation has been previously investigated for relationships with a variety of other environmental exposures. Methylation status was determined using methylation-specific quantitative PCR performed on genomic DNA extracted from whole blood. Results showed that relative methylation of IFN-γ promoter was significantly increased in DA+ in comparison with both comparator groups (DA- and AW), and it exhibited good sensitivity (77.5%) and specificity (80%) for identifying DA workers in a multivariate predictive model after adjusting for type of DI exposure, smoking status, methacholine PC₂₀, and gender. IL-4 promoter was slightly less methylated only in DA+ compared with AW among nonsmoking workers. Both GSTM1 and DUSP22 promoter methylations were found not associated with DA. Our finding suggests that exposure to occupational chemicals could play a heretofore undefined mechanistic role via epigenetic modification of specific genes in the promoter region.

Pathogenesis and Disease Mechanisms of Occupational Asthma

Occupational asthma (OA) is one of the most common forms of work-related lung disease in all industrialized nations. The clinical management of patients with OA depends on an understanding of the multifactorial pathogenetic mechanisms that can contribute to this disease. This article discusses the various immunologic and nonimmunologic mechanisms and genetic susceptibility factors that drive the inflammatory processes of OA.

Hexamethylene diisocyanate asthma is associated with genetic polymorphisms of CD14, IL-13, and IL-4 receptor α

To the Editor, Diisocyanates are among the most common causes of occupational asthma. However, susceptibility factors and immune biomarkers of diisocyanate asthma (DA) have not been clearly defined. For example, serum diisocyanate antigen specific IgE and IgG have been extensively investigated but these immunoassays lack diagnostic accuracy in identifying workers with confirmed DA1, 2, 3. Various genetic variants have been identified as risk factors for DA in association studies. Certain HLA class II alleles and SNPs of antioxidant enzymes (e.g., glutathione-s-transferases, N- acetyl transferases) have been associated with confirmed DA, although these findings have not yet been replicated in multiple populations4. In 2006, we first reported that DA confirmed by specific inhalation challenge (SIC) testing was significantly associated with cytokine genotype combinations of interleukin 4 receptor alpha (IL4RA), interleukin 13 (IL13) and CD14 single nucleotide polymorphisms (SNPs), but exclusively in hexamethylene diisocyanate (HDI) exposed workers5. In this report, we confirm the aforementioned genotype associations in an expanded group of workers with confirmed DA when compared to diisocyanate exposed workers without DA. A total of 368 diisocyanate-exposed workers were recruited by clinical investigators at four occupational disease clinics (Hopital du Sacre-Cœur de Montreal, Montreal, Canada; Hopital Laval, Sainte-Foy, Canada; University Health Network, Toronto, Canada; and Fundacion Jimenez Diaz, Madrid, Spain) and included: 103 diagnosed with DA (DA+) based on a positive SIC test; 115 symptomatic workers with negative SIC tests (DA−); and 150 HDI-exposed asymptomatic control spray paint workers (i.e, healthy subjects, not exhibiting respiratory symptoms). Blood samples were obtained for DNA extraction and genotyped for IL4RA (I50V), IL4RA (Q551R), IL4RA (E375A), IL13 (R110Q) and CD14 (C159T) SNPs, as previously described5. Subjects were predominantly male (91%), Caucasian (99%) and of European descent (90.8% French Canadian, 2.7% English Canadian, 1.9 % Spanish). Of 103 workers with DA, 50, 22, and 31 were exposed to HDI, MDI, and TDI, respectively, Of the 115 DA− symptomatic workers, 91, 18, and 6 were exposed to HDI, MDI, and TDI, respectively, while all asymptomatic controls were exposed to HDI. The duration of workplace exposure in months (mean ± SD) was 137 ± 13.9 for DA+, 157.9 ± 14.2 for DA−, and 65.6 ± 2.2 for asymptomatic HDI workers. All SNPs studied were in Hardy-Weinberg equilibrium (chi-square tests, all p>0.10). No significant associations were identified (by the chi-square test) between DA and individual alleles or genotypes of the candidate SNPs for IL4RA, IL13, or CD14. Genotypes were dichotomized for further statistical analyses as follows: IL4RA (I50V), II vs. IV or VV; IL4RA (Q551R), QQ vs. QR or RR; IL4RA (E375A), EE vs. EA or AA; IL-13(R110Q), RR vs. RQ or QQ; CD14 (C159T), CT vs. CC or TT. Combinations of genotypes were also dichotomized to compare the indicated combination to all other possible combinations. For example, in Table I, the combination of IL4RA II and IL13 RR compared IL4RA (I50V) = II and IL13 (R110Q) = RR to all other combinations of IL4RA (I50V) and IL-13(R110Q). Logistic regression analysis revealed significant interactions of diisocyanate exposure (HDI vs. MDI, TDI) with specific genotype combinations (i.e., IL4RA II + IL13RR; IL4RA II + CD14 CT; and IL4RA II + IL13 RR + CD14 CT) for distinguishing confirmed DA status (DA+) in comparison to SIC negative workers (DA−), after adjusting for significantly associated demographic variables (Table I). TABLE I Logistic regression analyses of associations of genotype or genotype combinations and exposure (HDI vs. MDI and/or TDI) with OA confirmed by specific inhalation challenge (DA+) versus specific inhalation challenge negative (DA−) workers in all ... In logistic regression analyses comparing HDI-exposed DA+ workers (n= 50) with HDI-exposed DA− workers (n = 91), DA remained significantly associated with IL4RA II + CD14 CT and IL4RA II + IL13 RR + CD14 CT genotype combinations (Table I) after adjustment for smoking status and gender. When comparing HDI-exposed DA+ workers (n= 50) with asymptomatic HDI-exposed workers (n = 150), the association between DA and the IL4RA II + CD14 CT and IL4RA II + IL13 RR + CD14 CT genotype combinations approached statistical significance (p < 0.10) after adjustment for age at diagnosis and smoking status (Table II). TABLE II Logistic regression analyses in HDI exposed workers of associations of genotype or genotype combinations with specific inhalation challenge positive workers (DA+) versus asymptomatic workers (Controls), after adjusting for significant demographic variables. ... These results in an expanded group of workers confirm our original findings of significant associations of DA with IL4RA (I50V), IL13 (R110Q) and CD14 (C159T) genotype combinations modified by exposure to HDI5. Unique to this report is the finding that two genotype combinations remain significantly associated with DA when compared with DA− workers and a similar, but not statistically significant, association was observed when compared with an asymptomatic cohort of HDI exposed workers. Significant associations between genotype and occupational asthma were found only after adjustment for work relevant diisocyanate exposure (i.e., HDI vs.TDI, MDI). The reason for restriction of this finding to the HDI exposed population is unknown and may be an artifact of the greater numbers of HDI-exposed subjects available for statistical analysis. This study employed a case control design using a candidate gene approach. The rarity of diisocyanate asthma and the relatively small numbers of subjects able to be recruited compared with genetic studies of non-occupational asthma is a limitation of this study and a hindrance to future replication studies. However, the issue of small group sizes could be counter-balanced by the ability to precisely define the DA phenotype using objective SIC tests with work-relevant diisocyanate chemicals. The role of these genotype combinations in the pathogenesis of DA is unknown and awaits functional characterization. Nevertheless, susceptibility genes associated with Th2 helper cell differentiation (e.g., IL4RA, IL13) and innate immunity (CD14) have been extensively investigated in non-occupational asthma6. The IL13 (R110Q) SNP has been associated with asthma and airway hyperresponsiveness and IL4RA variant V and R alleles (I50V and Q551R, respectively) have been associated with asthma and atopy7, 8. No such associations were detected in the present study of workers which were not enriched with atopic subjects. In summary, we have confirmed a previous observation in expanded groups of workers: a reported association between genotype combinations associated with Th2 and innate immunity and diisocyanate-induced asthma caused by HDI. Replication of these results in other background populations will be necessary to define the possible value of these genetic markers for risk assessment.

Exposure to Protein Aeroallergens in Egg Processing Facilities

Proteinaceous materials in the air can be highly allergenic and result in a range of immunologically mediated respiratory effects, including asthma. We report on the largest evaluation of exposure to date of airborne egg protein concentrations in an egg breaking and processing plant that had cases of occupational asthma. Personal air samples for egg protein were analyzed in duplicate on each PTFE filter using two analytical methods: (1) a commercial assay for non-specific total protein, and (2) indirect competitive inhibition assay using an ELISA method to quantify specific egg protein components. The highest concentrations were found in the egg washing room (mean exposure 644 microg/m3) and breaking room (255 microg/m3), which were also the areas where the risk of being sensitized was the greatest. There was excellent quantitative agreement between the airborne concentrations of total protein and sum of the specific protein antigens (ovalbumin, ovomucoid, and lysozyme). The correlation coefficient of the log-transformed data from the two methods was 0.88 (p < 0.0001). Size-selective sampling also indicated that most of the aerosol was capable of reaching the small airways. The methods described can be utilized to evaluate employee exposure to egg proteins. Exposure documentation, coupled with recommended exposure reduction strategies, could facilitate prevention of future employee sensitization and allergic respiratory responses by identifying high-exposure jobs and evaluating control measures.

A case of progesterone-induced anaphylaxis, cyclic urticaria/angioedema, and autoimmune dermatitis.

OBJECTIVE Women have exhibited anaphylaxis, urticaria/angioedema, and autoimmune progesterone dermatitis (APD) coinciding with the progesterone premenstrual rise. We report a detailed immunological evaluation of such a woman responsive to a gonadotropin hormone-releasing agonist (GHRA). METHODS Skin testing, enzyme-linked immunosorbent assays (ELISAs), leukocyte histamine release (LHR), and inhibition assays were performed to demonstrate progesterone immunoresponsiveness. RESULTS Serum specific-progesterone immunoglobulin G (IgG) and IgE were detected initially and disappeared 6 months after GHRA treatment. Dose-response LHR using patient basophils was observed for different hormones but after 3 months persisted only for 5β-pregnanediol. Preincubation with mouse antiprogesterone monoclonal antibody (PmAb) or mifepristone, a progesterone inhibitor, over a range of doses inhibited specific progesterone-induced LHR. Experiments with varying progesterone concentrations and a fixed dose of anti-IgE resulted in 100% LHR at a concentration as low as 0.016 nmol/mL, which, without anti-IgE, failed to release histamine. CONCLUSIONS This is the first report of combined recurrent anaphylaxis, cyclic urticaria/angioedema, and APD induced by immunoresponsiveness to progesterone.