Paul D. Siegel

Effect of diesel exhaust particulate (DEP) on immune responses: contributions of particulate versus organic soluble components

The effect of diesel exhaust particulate (DEP) exposure on innate, cellular and humoral pulmonary immunity was studied using high-dose, acute-exposure rat, mouse, and cell culture models. DEP consists of a complex mixture of petrochemical-derived organics adsorbed onto elemental carbon particles. DEP is a major component of particulate urban air pollution and a health concern in both urban and occupational environments. The alveolar macrophage is considered a key cellular component in pulmonary innate immunity. DEP and DEP organic extracts have been found to suppress alveolar macrophage function as demonstrated by reduced production of cytokines (interleukin-1 [IL-1], tumor necrosis factor-α[TNF-α]) and reactive oxygen species (ROS) in response to a variety of agents, including lipopolysaccharide (LPS), interferon-γ(IFN-γ), and bacteria. Fractionation of DEP organic extract suggests that this activity was predominately in polyaromatic-containing and more polar (resin) fractions. Organic-stripped DEP did not alter these innate pulmonary immune responses. DEP also depressed pulmonary clearance of Listeria monocytogenes and Bacillus Calmette-Guerin (BCG). The contribution of the organic component of DEP is less well defined with respect to acquired and humoral immunity. Indeed, both DEP and carbon black enhanced humoral immune responses (specific immunoglobulin [Ig] E and IgG) in an ovalbumin-sensitized rat model. It is concluded that both the particulate and adsorbed organics may contribute to DEP-mediated immune alterations.

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI-TOF and MALDI-qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H](+) ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI-MS; however, these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI-TOF MS.

The LLNA: A Brief Review of Recent Advances and Limitations

Allergic contact dermatitis is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. This highlights the importance of developing rapid and sensitive methods for hazard identification of chemical sensitizers. The murine local lymph node assay (LLNA) was developed and validated for the identification of low molecular weight sensitizing chemicals. It provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. However, there are also several concerns with this assay including: levels of false positive responses, variability due to vehicle, and predictivity. This report serves as a concise review which briefly summarizes the progress, advances and limitations of the assay over the last decade.

Haptenation: chemical reactivity and protein binding.

Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

A sensitive new bioassay for tumor necrosis factor

Tumor necrosis factor is an important cytokine involved in inflammation and assay of this cytokine in biological fluids may be important in the understanding of several disease processes. This report describes an improved TNF bioassay employing a newly isolated subclone of the cell line NCTC-clone 929 as well as a novel fluorescence indicator system for detecting viability of the target cells. The limit of detection for the TNF hypersensitive cell line with this fluorescence viability assay was 68 +/- 2.5 fg/ml, which is approximately 3 x more sensitive than the parental clone and approximately 10 x more sensitive than that reported by Branch et al. (1991) using the neutral red indicator system. The hypersensitivity of the clone gradually declined over a 45-day period and at regular intervals new cells were cultivated from frozen stocks. Two different serum sources, bovine fetal serum and horse serum, and four different serum concentrations (5, 10, 15, 20%) were evaluated to optimize sensitivity. No difference was found between serum sources but sensitivity was significantly reduced if < 15% serum was used.

Endotoxin Exposure and Inflammation Markers Among Agricultural Workers in Colorado and Nebraska

The adverse respiratory effects of agricultural dust inhalation are mediated in part by endotoxin, a constituent of gram-negative bacterial cell walls. This study quantified personal work-shift exposures to inhalable dust, endotoxin, and its reactive 3-hydroxy fatty acid (3-OHFA) constituents among workers in grain elevators, cattle feedlots, dairies, and on corn farms. Exposures were compared with post-work-shift nasal lavage fluid inflammation markers and respiratory symptoms. Breathing-zone personal air monitoring was performed over one work shift to quantify inhalable dust (Institute of Medicine samplers), endotoxin (recombinant factor C [rFC] assay), and 3-OHFA (gas chromatography/mass spectrometry). Post-shift nasal lavage fluids were assayed for polymorphonuclear neutrophils (PMN), myeloperoxidase (MPO), interleukin 8 (IL-8), albumin, and eosinophilic cation protein (ECP) concentrations. The geometric mean (GSD) of endotoxin exposure (rFC assay) among the 125 male participants was 888 ± (6.5) EU/m3, and 93% exceeded the proposed exposure limit (50 EU/m3). Mean PMN, MPO, albumin, and ECP levels were two- to threefold higher among workers in the upper quartile of 3‐OHFA exposure compared to the lowest exposure quartile. Even numbered 3-OHFA were most strongly associated with nasal inflammation. Symptom prevalence was not elevated among exposed workers, possibly due to endotoxin tolerance or a healthy worker effect in this population. This is the first study to evaluate the relationship between endotoxins 3-OHFA constituents in agricultural dust and nasal airway inflammation. More research is needed to characterize the extent to which these agents contribute to respiratory disease among agricultural workers.

Acute Upper and Lower Respiratory Effects in Wildland Firefighters

Objectives: To assess acute respiratory effects experienced by wildland firefighters. Methods: We studied two Interagency Hotshot Crews with questionnaires, spirometry, and measurement of albumin, eosinophilic cationic protein (ECP), and myeloperoxidase (MPO) as indicators of inflammation in sputum and nasal lavage fluid. Assessments were made preseason, postfire, and postseason. Results: Fifty-eight members of the two crews had at least two assessments. Mean upper and lower respiratory symptom scores were higher postfire compared to preseason (P < 0.001). The mean forced expiratory volume in 1 second was lower postfire compared to preseason (P < 0.001) and then recovered by postseason. Individual increases in sputum and nasal ECP and MPO from preseason to postfire were all significantly associated with postfire respiratory symptom scores. Conclusions: Wildland firefighting was associated with upper and lower respiratory symptoms and reduced forced expiratory volume in 1 second. Within individuals, symptoms were associated with increased ECP and MPO in sputum and nasal lavage fluid. The long-term respiratory health impact of wildland firefighting, especially over multiple fire seasons, remains an important concern.

THE EFFECT OF DIESEL EXHAUST PARTICLES (DEP) AND CARBON BLACK (CB) ON THIOL CHANGES IN PULMONARY OVALBUMIN ALLERGIC SENSITIZED BROWN NORWAY RATS

Brown Norway rats were exposed by intratracheal instillation of saline, carbon black (CB), or diesel exhaust particles (DEP) (5 mg/kg) on day 1, followed by exposure to ovalbumin (OVA, 90 mg/m 3) or saline for 30 minutes on days 1, 8, 15, and 29. Animals were sacrificed on day 30. The DEP, CB, or OVA exposure alone did not result in abnormal levels of inflammatory cells, lactate dehydrogenase (LDH), or total protein in the lavage fluid. In combined OVA-DEP or OVA-CB exposure, however, these markers were significantly increased. The adjuvant effect of CB and DEP on OVA sensitization was evidenced by the marked increases in serum OVA-specific IgG (5.6-fold) and IgE (3.5-4 fold) levels, and the increase in interleukin-4 (IL-4) mRNA levels in lung tissue. The OVA exposure markedly reduced glutathione (GSH) levels in both cell types. In combined DEP-OVA exposure, the level of GSH in lymphocytes was further decreased, indicating a possible interactive effect between DEP and OVA exposures. These results show that both DEP and CB augmented OVA-induced allergic sensitization, and that particle composition of DEP may not be a critical factor for the adjuvant effect. OVA exposure causes significant depletion of intracellular GSH in lymphocytes, which may play a key role in OVA-mediated immune responses.

Rapid and simple kinetics screening assay for electrophilic dermal sensitizers using nitrobenzenethiol.

The need for alternatives to animal-based skin sensitization testing has spurred research on the use of in vitro, in silico, and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped-flow techniques and conventional UV spectrophotometric measurements enabled the determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for seven extreme, five strong, seven moderate, and four weak/nonsensitizers. Seventeen out of the 23 tested chemicals were pseudo-first order, and three were second order. In three out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate, and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, select diones, and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid, and inexpensive absorbance-based method has great potential for use as a preliminary screening tool for skin allergens.

Allergic Contact Dermatitis to Synthetic Rubber Gloves: Changing Trends in Patch Test Reactions to Accelerators

BACKGROUND Rubber gloves are one of the most frequent causes of occupational allergic contact dermatitis, especially in health care workers. OBSERVATIONS We describe 23 patients with allergic contact dermatitis due to rubber accelerators in rubber gloves, some with disseminated dermatitis, treated during a 2-year period. Three had IgE-mediated latex allergies. Sixteen were health care workers from a single institution whose dermatitis was temporally related to the switch to latex-safe gloves. Each had positive patch test reactions to 1 or more rubber accelerators, including carbamates, thiurams, 2-mercaptobenzothiazole, and 1,3-diphenylguanidine. Chemical analysis of 6 glove samples identified 2-mercaptobenzothiazole in 4 and zinc diethyldithiocarbamate in 1. There were discordances between patch test results for glove chemicals and glove swatches and between available information on chemicals used during glove production and chemicals detected during glove analysis. Although these factors may complicate the search for culprit and alternative gloves, dermatitis cleared in each of 9 patients with follow-up data and for whom alternative gloves were provided based on published information of glove composition. CONCLUSIONS Allergic contact dermatitis due to synthetic rubber gloves occurs even with the use of latex-safe products. More knowledge about chemicals present in these gloves, to which the skin is exposed during use, is necessary to prevent and treat allergic contact dermatitis.