Peter S. Thorne

CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract.

Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.

Bioaerosol Concentrations in Noncomplaint, Complaint, and Intervention Homes in the Midwest

Forty-one homes of conventional design were studied to investigate the relationship between bioaerosols, building parameters, and season, and to determine if differences existed across health-based home categories. The homes were categorized as: those for which no indoor air problems were known (noncomplaint homes), noncomplaint homes of allergy patients (intervention homes), and complaint “sick” homes (complaint homes). Carbon dioxide and relative humidity were measured in the basement and main floor areas. CO2 concentrations were elevated for complaint homes (mean 1190 ppm) but less than 1000 ppm for all noncomplaint homes (mean 550 ppm). Relative humidity was significantly lower for intervention homes than for complaint or noncomplaint homes. Viable and nonviable bioaerosol sampling was performed on the main floor, the basement, and outside. Outdoor viable fungi exhibited an 8.4-fold range when plotted by month, but respirable and nonrespirable indoor fungal concentrations did not differ significantly ...

Increased Levels of Markers of Microbial Exposure in Homes with Indoor Storage of Organic Household Waste

ABSTRACT As part of environmental management policies in Europe, separate collection of organic household waste and nonorganic household waste has become increasingly common. As waste is often stored indoors, this policy might increase microbial exposure in the home environment. In this study we evaluated the association between indoor storage of organic waste and levels of microbial agents in house dust. The levels of bacterial endotoxins, mold β(1→3)-glucans, and fungal extracullar polysaccharides (EPS) of Aspergillus andPenicillium species were determined in house dust extracts as markers of microbial exposure. House dust samples were collected in 99 homes in The Netherlands selected on the basis of whether separated organic waste was present in the house. In homes in which separated organic waste was stored indoors for 1 week or more the levels of endotoxin, EPS, and glucan were 3.2-, 7.6-, and 4.6-fold higher, respectively (all P < 0.05), on both living room and kitchen floors than the levels in homes in which only nonorganic residual waste was stored indoors. Increased levels of endotoxin and EPS were observed, 2.6- and 2.1-fold (P < 0.1), respectively, when separated organic waste was stored indoors for 1 week or less, whereas storage of nonseparated waste indoors had no effect on microbial agent levels (P > 0.2). The presence of textile floor covering was another major determinant of microbial levels (P < 0.05). Our results indicate that increased microbial contaminant levels in homes are associated with indoor storage of separated organic waste. These increased levels might increase the risk of bioaerosol-related respiratory symptoms in susceptible people.

Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-Sampling Methods

ABSTRACT Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using theLimulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.

Contact sensitivity by diisocyanates: Potencies and cross-reactivities

The dermal sensitizing potencies of four diisocyanates were investigated using the mouse ear-swelling test (MEST) with BALB/cBy mice. Mice were administered topical doses of diisocyanates to the abdomen and were challenged on the ear 4 days later with a nonirritating dose of chemical. The increase in ear thickness at 24 hr postchallenge indicated the extent of contact sensitivity. Ear thickness increase plotted against the log of the dose of diisocyanate indicated three regions of effects: a no-effect region, a dose-response region, and a region of reduced response at highest dosages. The potencies of the diisocyanates expressed as the SD50 (dose required to sensitize 50% of the animals in each group) were hexamethylene diisocyanate, 0.088 mg/kg; diphenylmethane-4,4-diisocyanate, 0.73 mg/kg; and toluene diisocyanate, 5.3 mg/kg. For dicyclohexylmethane diisocyanate, the steep dose-response curve suggested a threshold-type response occurring at 0.24 mg/kg. The specificity of sensitization was evaluated by challenging sensitized animals with heterologous diisocyanates. The homologous reactions were always the most extensive. However, cross-reactions were noted between aryl and alkyl diisocyanates. Toluene diisocyanate, the weakest sensitizer, elicited the fewest cross-reactions. The MEST proved to be a simple and effective method for assessing delayed-type hypersensitivity without need of adjuvants, occlusive patches, abrasions, or other procedures which fail to mimic industrial exposures. The observation that very high exposures resulted in reduced response indicates that protocols which generate dose-response data must be utilized to accurately assess the sensitizing potencies of industrial chemicals.

Field Evaluation of Endotoxin Air Sampling Assay Methods

This study tested the importance of filter media, extraction and assay protocol, and bioaerosol source on the determination of endotoxin under field conditions in swine and poultry confinement buildings. Multiple simultaneous air samples were collected using glass fiber (GF) and polycarbonate (PC) filters, and these were assayed using two methods in two separate laboratories: an endpoint chromogenic Limulus amebocyte lysate (LAL) assay (QCL) performed in water and a kinetic chromogenic LAL assay (KQCL) performed in buffer with resistant-parallel line estimation analysis (KLARE). In addition, two aqueous filter extraction methods were compared in the QCL assay: 120 min extraction at 22 degrees C with vigorous shaking and 30 min extraction at 68 degrees C with gentle rocking. These extraction methods yielded endotoxin activities that were not significantly different and were very highly correlated. Reproducibility of endotoxin determinations from duplicate air sampling filters was very high (Cronbach alpha all > 0.94). When analyzed by the QCL method GF filters yielded significantly higher endotoxin activity than PC filters. QCL and KLARE methods gave similar estimates for endotoxin activity from PC filters; however, GF filters analyzed by the QCL method yielded significantly higher endotoxin activity estimates, suggesting enhancement of the QCL assay or inhibition of the KLARE asay with GF filters. Correlation between QCL-GF and QCL-PC was high (r = 0.98) while that between KLARE-GF and KLARE-PC was moderate (r = 0.68). Analysis of variance demonstrated that assay methodology, filter-type, barn-type, and interactions between assay and filter-type and between assay and barn-type were important factors influencing endotoxin exposure assessment.

Respiratory symptoms and lung function abnormalities among machine operators in automobile production

This cross-sectional study was designed to assess differences in prevalence of respiratory symptoms and lung function between machine operators exposed to semisynthetic or soluble metal-working fluids (MWFs) and unexposed assemblers and to assess exposure-response relationships with MWF type, total aerosol, endotoxin, culturable bacteria and fungi. We evaluated 183 machine operators and 66 assemblers from one large automobile transmission plant using questionnaires, spirometry data, and cross-shift assessment of both lung function and respiratory symptoms. We found that airborne exposures to total aerosol, endotoxin, culturable bacteria and fungi were higher in machine operations than in the assembly area. There was a correlation between bulk and airborne culturable bacteria, but not between bulk and airborne culturable fungi. Machine operators had significantly more usual cough, usual phlegm, work-related chest tightness and post-shift symptoms of chest tightness, throat irritation, and cough compared with assemblers. We found exposure-response relationships between respiratory symptoms and total aerosol, as well as culturable fungi and bacteria. Associations with endotoxin were not strong or consistent, possibly because airborne levels were generally low. Cross-shift lung function decrements did not differ between machine operators and assemblers and there were no associations with MWF or specific exposures. The finding of respiratory symptoms at low levels of exposure in this study suggests the need to re-assess total aerosol thresholds. Associations between airborne fungal exposures and respiratory symptoms need further study to characterize sources of exposure other than MWF in machining operations.

Dermatitis among automobile production machine operators exposed to metal‐working fluids

This cross-sectional study was designed to assess differences in prevalence of contact dermatitis between machine operators exposed to metal-working fluids (MWFs) and unexposed assemblers, and to assess potential risk factors for contact dermatitis among these machine operators. In their work, machine operators were exposed to either semisynthetic or soluble oil MWFs. We evaluated 158 machine operators and 51 assemblers from one large automobile transmission plant using questionnaires, dermatologist examination of the skin, and dermal dosimetry to measure wetness and metal exposures. We found that machine operators had more combined (definite plus possible) dermatitis (27.2% vs. 13.7%, chi(2) = 3.9, p = 0.05, 1 df) compared with assemblers. Among machine operators, risk factors significantly associated with (combined) dermatitis were subjective assessment of wetness of the work, exposure to semisynthetic as opposed to soluble oil MWF, current cigarette smoking, and increasing worker age. These risk factors suggested preventive and control measures including control of wet work, surveillance program including early self-report of dermatitis, consideration of replacement of semisynthetic with soluble oil MWFs, and strictly limiting smoking among machine operators exposed to MWFs.

Assessment of airway reactivity in guinea pigs: Comparison of methods employing whole body plethysmography

Airway reactivity of guinea pigs was assessed while animals were in whole body plethysmographs by measurement of responses to histamine aerosol. Histamine was delivered using 2 protocols: one employed exposure to a single concentration, the other utilized 1.5-fold stepped concentrations. Both protocols were tested while animals inhaled ambient air, and again when the air contained 10% CO2. Exposures to single concentrations of histamine in air produced increases in both respiratory frequency (f) and plethysmograph pressure (delta P). The latter increase was shown to coincide with a decrease in tidal volume and with the onset of airway constriction. The time to reach a doubling in delta P represented a quantitative measure of airway reactivity. Exposures using the single concentration method in conjunction with 10% CO2 also demonstrated a dose-dependent time to reach the endpoint, in this case a 33% decrease in delta P from the maximum CO2-induced increase. The endpoints for the stepped concentration protocols were the concentration required to double delta P (PC2.delta P) or, in the presence of 10% CO2, the concentration that induced a 33% decline from the maximum delta P (PC33 (CO2]. Mean values for PC2.delta P and PC33 (CO2) were 2.1 mg/m3 and 0.58 mg/m3, respectively. These endpoints were comparable based on the dose received as calculated from the minute volume of animals when breathing the above atmospheres. The intersubject variability, assessed using both stepped concentration protocols, was comparable to that reported for PC20 FEV1 in human studies. Both stepped concentration protocols, conducted in air, or the 10% CO2 atmospheres, proved suitable for determination of airway reactivity of the unrestrained, unanesthetized guinea pigs. However, by not requiring use of CO2, the histamine in air approach is easier to perform, requires less time, and provides a clear endpoint representing early airway constriction.

Assessment of Particulates and Bioaerosols in Eastern Canadian Sawmills

The purpose of this study was to quantify and identify the airborne contamination in eastern Canadian sawmills. Seventeen sawmills were chosen to cover a wide range of size, geographic distribution, and wood species processed. Within each sawmill different work sites (debarking, sawing, sorting, or planing) were studied separately. Area sampling was performed for exposure assessment. Microbial contaminants were assessed with all-glass impingers 30 and six-stage Andersen microbial samplers; appropriate selective media and culture conditions for bacteria, thermophilic actinomycetes, molds, and yeasts were used. Inhalable dust, endotoxins, temperature, and humidity also were measured. Penicillium species were the most predominant molds with up to 40 different Penicillium species identified. Debarking was the working site most highly contaminated by molds, bacteria, and endotoxins (p=0.0001). At this working site mold levels reached a maximum of 1.5 x 10(6) CFU/m3, whereas the median values for culturable bacteria and endotoxin were 21,620 CFU/m3 and 1,081 endotoxin units/m3, respectively. Planing sites were the most highly dust contaminated (median: 3.0 mg/m3) (p <0.05). Sawmills of eastern Canada contain airborne biological contaminants that vary between working sites, and their microflora is different from that previously described in European sawmills.