Williamsia aurantiacus sp. nov. a novel actinobacterium producer of antimicrobial compounds isolated from the marine sponge.

An antibiotic-producing actinobacterium, designated isolate B375T, was isolated from marine sponge Glodia corticostylifera collected from Praia Guaecá, São Paulo, Brazil (23°49S; 45°25W), and its taxonomic position established using data from a polyphasic study. The organism showed a combination of morphological, physiological, biochemical and chemotaxonomic characteristics consistent with its classification in the genus Williamsia. Comparative 16S rRNA gene sequence analysis indicated that the strain B375T was most closely related to Williamsia serinedens DSM 45037T and Williamsia spongiae DSM 46676T and having 99.43% and 98.65% similarities, respectively, but was distinguished from these strains by a low level of DNA-DNA relatedness (53.2-63.2%) and discriminatory phenotypic properties. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and N-glycolated muramic acid residues present in the wall cells. The cells contained C16:0 (23.3%), C18:0 10-methyl (23.2%) and C18:1 ω9c (21.6%) as the major cellular fatty acids. The strain B375T inhibited growing of Staphylococcus aureus and Colletotrichum gloeosporioides strains and was considered a producer of antimicrobial compounds. Based on the data obtained, the isolate B375T (= CBMAI 1090T = DSM 46677T) should, therefore, be classified as the type strain of a novel species of the genus Williamsia, for which the name Williamsia aurantiacus sp. nov. is proposed.

Inflammatory potential in relation to the microbial content of settled dust samples collected from moisture-damaged and reference schools: results of HITEA study.

Aiming to identify factors causing the adverse health effects associated with moisture-damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture-damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n = 25) collected from moisture-damaged and reference schools in Spain, the Netherlands, and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor-α (TNF-)α, interleukin (IL)-6, and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis, and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers such as ergosterol, 3-hydroxy fatty acids, muramic acid, endotoxins, and glucans were measured as markers of exposure. Dust from moisture-damaged schools in Spain and the Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL-6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram-positive bacteria.

The use of multivariate curve resolution methods to improve the analysis of muramic acid as bacterial marker using gas chromatography-mass spectrometry: an alternative method to gas chromatography-tandem mass spectrometry.

In analysis of muramic acid (MA) as bacterial marker, two dominant disturbing factors lead the researchers to use gas chromatography-tandem mass spectrometry (GC-MS/MS) technique instead of gas chromatography-mass spectrometry (GC-MS). These factors are the trace concentration of MA and fundamental disturbance of base line mass channels in GC-MS technique. This study aimed to utilize multivariate curve resolution (MCR) methods combined with GC-MS to improve the analysis of MA. First, the background and noise in GC-MS analysis were corrected and reduced using MCR methods. In addition, the MA overlapped peaks were resolved to its pure chromatographic and mass spectral profiles. Then the two-way response of each component was reconstructed by the outer product of the pure chromatographic and mass spectral profiles. The overall volume integration (OVI) method was used for quantitative determination. The MA peak area was decreased dramatically after the background correction and noise reduction. The findings severely ratify the appropriateness of using MCR techniques combined with GC-MS analysis as a simple, fast and inexpensive method for the analysis of MA in complex mixtures. The proposed method may be considered as an alternative method to GC-MS/MS for thorough analysis of the bacterial marker.

Muramic acid, endotoxin, 3-hydroxy fatty acids, and ergosterol content explain monocyte and epithelial cell inflammatory responses to agricultural dusts.

In agricultural and other environments, inhalation of airborne microorganisms is linked to respiratory disease development. Bacterial endotoxins, peptidoglycans, and fungi are potential causative agents, but relative microbial characterization and inflammatory comparisons amongst agricultural dusts are not well described. The aim of this study was to determine the distribution of microbial endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, and ergosterol and evaluate inflammatory responses in human monocytes and bronchial epithelial cells with various dust samples. Settled surface dust was obtained from five environments: swine facility, dairy barn, grain elevator, domestic home (no pets), and domestic home with dog. Endotoxin concentration was determined by recombinant factor C (rFC). 3-OHFA, muramic acid, and ergosterol were measured using gas chromatography-mass spectrometry. Dust-induced inflammatory cytokine secretion in human monocytes and bronchial epithelial cells was evaluated. Endotoxin-independent dust-induced inflammatory responses were evaluated. Endotoxin and 3-OHFA levels were highest in agricultural dusts. Muramic acid, endotoxin, 3-OHFA, and ergosterol were detected in dusts samples. Muramic acid was highest in animal farming dusts. Ergosterol was most significant in grain elevator dust. Agricultural dusts induced monocyte tumor necrosis factor (TNF) alpha, interleukin (IL)-6, IL-8, and epithelial cell IL-6 and IL-8 secretion. Monocyte and epithelial IL-6 and IL-8 secretion was not dependent on endotoxin. House dust(s) induced monocyte TNFalpha, IL-6, and IL-8 secretion. Swine facility dust generally produced elevated responses compared to other dusts. Agricultural dusts are complex with significant microbial component contribution. Large animal farming dust(s)-induced inflammation is not entirely dependent on endotoxin. Addition of muramic acid to endotoxin in large animal farming environment monitoring is warranted.

Rubritalea spongiae sp. nov. and Rubritalea tangerina sp. nov., two carotenoid- and squalene-producing marine bacteria of the family Verrucomicrobiaceae within the phylum 'Verrucomicrobia', isolated from marine animals.

Two Gram-negative, non-motile, coccoid or rod-shaped, chemoheterotrophic bacteria designated strains YM21-132(T) and YM27-005(T) were isolated from marine animals, and were subjected to a polyphasic taxonomic examination. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the two isolates belong to the genus Rubritalea of the phylum 'Verrucomicrobia' (subdivision 1). The novel isolates shared approximately 97-98 % sequence similarity with each other and showed 93-97 % similarity with Rubritalea species of the family Verrucomicrobiaceae. The level of DNA-DNA relatedness between strains YM21-132(T) and YM27-005(T) was less than 70 %, which is accepted as the phylogenetic definition of a species. Both strains produced reddish carotenoid pigments and squalene. The cell wall peptidoglycan of both strains contained muramic acid and meso-diaminopimelic acid. The G+C contents of the genomic DNA were 48.0 mol% (strain YM21-132(T)) and 50.3 mol% (strain YM27-005(T)). The presence of MK-8 and MK-9 as the major isoprenoid quinones, and iso-C(14 : 0), iso-C(16 : 0) and C(16 : 1)omega7c as the major cellular fatty acids supported the identification of the two novel strains as members of the genus Rubritalea. On the basis of polyphasic taxonomic studies, it was concluded that these strains should be classified as representing two novel, separate species in the genus Rubritalea within the phylum 'Verrucomicrobia', for which the names Rubritalea spongiae sp. nov. (type strain YM21-132(T)=MBIC08281(T)=KCTC 12906(T)) and Rubritalea tangerina sp. nov. (type strain YM27-005(T)=MBIC08282(T)=KCTC 12907(T)) are proposed.

Cerasicoccus arenae gen. nov., sp. nov., a carotenoid-producing marine representative of the family Puniceicoccaceae within the phylum 'Verrucomicrobia', isolated from marine sand.

A polyphasic taxonomic study was performed on strain YM26-026(T), which was isolated from acid-treated sediment in Kamaishi, Japan. The bacterial cells were pale-pink-pigmented, Gram-negative, obligately aerobic, non-spore-forming, spherical and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel isolate was a member of the phylum 'Verrucomicrobia' and shared approximately 84-87 % sequence similarity with strains of the class Opitutae that have been cultivated to date. Strain YM26-026(T) produced pale-pink pigments of carotenoid. beta-Lactam antibiotic susceptibility tests and amino acid analysis of cell-wall hydrolysates indicated that the novel isolate did not contain muramic acid or diaminopimelic acid in the cell wall, suggesting that the strain lacks peptidoglycan. The G+C content of the DNA of strain YM26-026(T) was 54.0 mol%. Menaquinone-7 was the major quinone and C(14 : 0) and C(18 : 1)omega9c were the major fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that strain YM26-026(T) represents a new genus of the family Puniceicoccaceae within the phylum 'Verrucomicrobia', for which the name Cerasicoccus arenae gen. nov., sp. nov. is proposed. The type strain is YM26-026(T) (=MBIC08280(T)=KCTC 12870(T)).

Pelagicoccus mobilis gen. nov., sp. nov., Pelagicoccus albus sp. nov. and Pelagicoccus litoralis sp. nov., three novel members of subdivision 4 within the phylum 'Verrucomicrobia', isolated from seawater by in situ cultivation.

Five Gram-negative, white-pigmented, spherical, chemoheterotrophic bacteria were isolated from seawater from Japan and the Republic of Palau by use of an in situ cultivation technique. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the five novel isolates, 02PA-Ca-133(T), YM14-201(T), H-MN57(T), H-MN48 and MN1-156, were closely affiliated to members of subdivision 4 within the phylum 'Verrucomicrobia'. The novel isolates shared 96-100 % sequence similarity with each other and showed less than 90 % similarity with the cultivated strains of subdivision 4. DNA-DNA relatedness values between strains 02PA-Ca-133(T), YM14-201(T) and H-MN57(T) were less than 70 %; the value commonly accepted as the threshold for the phylogenetic definition of a species. Antibiotic susceptibility tests and amino acid analysis of cell-wall hydrolysates indicated that the novel isolates did not contain muramic acid or diaminopimelic acid in their cell walls, suggesting that these strains lack peptidoglycan. The DNA G+C contents of the five strains were 51-57 mol%. The major menaquinone was MK-7 and C(16 : 0), C(16 : 1)omega 7c and anteiso-C(15 : 0) were the major fatty acids. On the basis of polyphasic taxonomic evidence, it is concluded that these strains should be classified as representing a new genus and three novel species in subdivision 4 of the phylum 'Verrucomicrobia', for which the names Pelagicoccus mobilis gen. nov., sp. nov. [type strain 02PA-Ca-133(T) (=MBIC08004(T)=IAM 15422(T)=KCTC 13126(T))], Pelagicoccus albus sp. nov. [type strain YM14-201(T) (=MBIC08272(T)=IAM 15421(T)=KCTC 13124(T))] and Pelagicoccus litoralis sp. nov. [type strain H-MN57(T) (=MBIC08273(T)=IAM 15423(T)=KCTC 13125(T))] are proposed.

Coraliomargarita akajimensis gen. nov., sp. nov., a novel member of the phylum 'Verrucomicrobia' isolated from seawater in Japan.

An obligately aerobic, Gram-negative, non-spore-forming, non-motile, spherical bacterium, designated strain 04OKA010-24(T), was isolated from seawater surrounding the hard coral Galaxea fascicularis L., collected at Majanohama, Akajima, Japan, and was subjected to a polyphasic taxonomic study. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new strain represented a member of the phylum 'Verrucomicrobia' and shared 84-95 % sequence similarity with cultivated strains of 'Verrucomicrobia' subdivision 4. Amino acid analysis of the cell-wall hydrolysate indicated the absence of muramic acid and diaminopimelic acid, which suggested that the strain did not contain peptidoglycan in the cell wall. The G+C content of the DNA was 53.9 mol%. MK-7 was the major menaquinone and C(14 : 0), C(18 : 1)omega9c and C(18 : 0) were the major fatty acids. On the basis of these data, it was concluded that strain 04OKA010-24(T) represents a novel species in a new genus in subdivision 4 of the phylum 'Verrucomicrobia', for which the name Coraliomargarita akajimensis gen. nov., sp. nov. is proposed. The type strain of Coraliomargarita akajimensis is 04OKA010-24(T) (=MBIC06463(T)=IAM 15411(T)=KCTC 12865(T)).

Gordonia soli sp. nov., a novel actinomycete isolated from soil.

A soil isolate, strain CC-AB07T, was characterized using a polyphasic approach. This organism had chemotaxonomic and morphological properties consistent with its classification in the genus Gordonia. 16S rRNA gene sequence analysis showed that the novel strain formed a monophyletic branch at the periphery of the evolutionary radiation occupied by the genus Gordonia, its closest neighbours being the type strains of Gordonia alkanivorans, Gordonia amicalis, Gordonia bronchialis, Gordonia desulfuricans, Gordonia polyisoprenivorans and Gordonia rhizosphera. The novel isolate was distinguished from all of these type strains using a range of phenotypic properties and by gyrB gene sequence analysis. It was evident from the genotypic and phenotypic data that strain CC-AB07T should be classified as representing a novel species in the genus Gordonia, for which the name Gordonia soli sp. nov. is proposed. The type strain is CC-AB07T (=BCRC 16810T=DSM 44995T).

Actinocatenispora thailandica gen. nov., sp. nov., a new member of the family Micromonosporaceae.

Two actinomycete strains, TT2-10(T) and TT2-3, which produced long spore chains (more than 10 spores per chain), were isolated from peat swamp forest soil in Pattaloong Province, Thailand. Their taxonomic positions were determined using a polyphasic approach. The chemotaxonomic characteristics of these strains coincided with those of the family Micromonosporaceae, i.e. cell-wall chemotype II, muramic acid of the N-glycolyl type, whole-cell sugar pattern D and type II phospholipids. Analysis of the 16S rRNA gene sequences also indicated that these strains constitute a distinct lineage within the family Micromonosporaceae, sharing 91.3-93.8 % sequence similarity with members of this family. On the basis of their phenotypic and genotypic characteristics and their phylogenetic position, these strains represent a novel genus and species, for which the name Actinocatenispora thailandica gen. nov., sp. nov. is proposed. The type strain of Actinocatenispora thailandica is strain TT2-10(T) (=JCM 12343(T)=PCU 235(T)=DSM 44816(T)).

On-line sample preconcentration with chemical derivatization of bacterial biomarkers by capillary electrophoresis: a dual strategy for integrating sample pretreatment with chemical analysis.

Simple, selective yet sensitive methods to quantify low-abundance bacterial biomarkers derived from complex samples are required in clinical, biological, and environmental applications. In this report, a new strategy to integrate sample pretreatment with chemical analysis is investigated using on-line preconcentration with chemical derivatization by CE and UV detection. Single-step enantioselective analysis of muramic acid (MA) and diaminopimelic acid (DAP) was achieved by CE via sample enrichment by dynamic pH junction with ortho-phthalaldehyde/N-acetyl-L-cysteine labeling directly in-capillary. The optimized method resulted in up to a 100-fold enhancement in concentration sensitivity compared to conventional off-line derivatization procedures. The method was also applied toward the detection of micromolar levels of MA and DAP excreted in the extracellular medium of Escherichia coli bacterial cell cultures. On-line preconcentration with chemical derivatization by CE represents a unique approach for conducting rapid, sensitive, and high-throughput analyses of other classes of amino acid and amino sugar metabolites with reduced sample handling, where the capillary functions simultaneously as a concentrator, microreactor, and chiral selector.

Bacterial components in the synovial tissue of patients with advanced rheumatoid arthritis or osteoarthritis: analysis with gas chromatography-mass spectrometry and pan-bacterial polymerase chain reaction.

OBJECTIVE: To study the presence of bacterial components in the synovial tissue (ST) of patients with advanced rheumatoid arthritis (RA). METHODS: ST was collected during joint surgery from 41 RA patients. Tissue from 39 patients with osteoarthritis (OA), 4 patients with undifferentiated inflammatory arthritis (UA), and 3 cases of accidental deaths served as controls. The pan-bacterial polymerase chain reaction (PCR) with primers for the 23S ribosomal RNA (rRNA) and 16S rRNA genes was used to detect bacterial DNA. In addition, synovial fluid (SF) samples from patients with chlamydial reactive arthritis (ReA) were also examined by the same method. The positive controls, bacterial DNA or ST spiked with different living bacteria, were analyzed alongside clinical samples. Most of the ST samples were also analyzed by gas chromatography-mass spectrometry (GC-MS) for determining the presence of bacteria-derived muramic acid. Strict precautions were followed in the clinics and the laboratory to prevent contamination. RESULTS: In GC-MS analysis, muramic acid was observed in the ST from 4 of 35 RA patients and from 2 of 14 OA patients, but not in ST from 2 patients with UA and 3 cadavers. Bacterial DNA was not detected by either one of the PCR primers used in ST from 42 patients with RA and 39 patients with OA. However, 5 of 15 SF samples from ReA patients were PCR positive. The sensitivity of GC-MS to detect muramic acid was 2 pg/injected amount (227 pg muramic acid/mg ST), and that of the pan-bacterial PCR was 2-20 bacteria colony forming units/reaction. CONCLUSION: These results indicate that a bacterial component, muramic acid, is detectable by GC-MS in ST from a few patients with advanced RA or OA. However, no bacterial DNA was detectable by PCR.

Analysis of a stable halogenated derivative of muramic acid by gas chromatography-negative ion chemical ionization tandem mass spectrometry.

Muramic acid (Mur) is present in the cell wall of Eubacteria and serves as a chemical marker for the trace detection of bacteria and bacterial cell wall debris in complex matrices. There have been numerous studies using a variety of derivatives of Mur, particularly in combination with gas chromatography-tandem mass spectrometry (GC-MS-MS) where the detection limit has been steadily lowered. A stable, halogenated derivative, the pentafluorobenzyl oxime (PFBO) acetate of Mur, has been developed by others and successfully used for GC with electron-capture detection. The current report is the first use of this derivative for GC-MS-MS analysis of Mur, or indeed any other carbohydrate, using negative ion chemical ionization (NICI) with GC-MS-MS. Mur was readily detected in settled surface dust (166 ng/mg), as well as dust collected from indoor air (1.4-5.9 ng/mg). Analyses of Mur as a PFBO acetate by GC-NICI-MS-MS or as alditol acetates by electron impact GC-electron impact ionization MS-MS serve as complementary approaches for trace detection in complex matrices.

Muramic acid is not generally present in the human spleen as determined by gas chromatography-tandem mass spectrometry.

It has been hypothesized that bacterial debris may accumulate in tissues of the reticuloendothelial system (RES) serving as an inflammatory stimulus for human disease. In support of this hypothesis, muramic acid (Mur), a component of bacterial peptidoglycan (PG), has previously been reported to be present in culture-negative human spleen. High-performance liquid chromatography (HPLC) was employed in these analyses, and a peak was detected at the retention time of Mur. However, HPLC is best used as a screening technique, and it is vital that these tentative observations be reexamined by the state-of-the-art approach (gas chromatography-tandem mass spectrometry [GC-MS(2)]). Indeed, in the present work using GC-MS(2), Mur was not detected in six out of seven human spleens previously examined by HPLC. However, Mur was categorically detected at minute concentrations, 50 ppb, in one spleen. In conclusion, since Mur is not generally found in culture-negative human spleen, in future studies, these tissues can serve as negative controls. The study of Mur levels in inflammation (e.g., reactive arthritis) could prove important in testing the hypothesis that bacterial debris persisting in tissues could serve as a depot inciting diseases of unknown etiology.

Enzyme degradation and proinflammatory activity in arthritogenic and nonarthritogenic Eubacterium aerofaciens cell walls.

Two almost-identical strains of Eubacterium aerofaciens isolated from the normal human gut flora were used. The cell wall (CW) of one strain with a peptidoglycan (PG) type A4alpha induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4beta induces only a transient acute arthritis. The CW of the arthritogenic E. aerofaciens was a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW. After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-alpha and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased. In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG. These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines.

New and simple procedure for the determination of muramic acid in chemically complex environments by gas chromatography-ion trap tandem mass spectrometry.

A gas chromatographic-ion trap tandem mass spectrometric method was developed for the quantification of muramic acid, a marker of bacterial peptidoglycan, in environmental and clinical specimens. Samples (bacteria, house dust and urine) were heated in methanolic hydrochloric acid overnight and extracted with hexane for removal of hydrophobic compounds. The aqueous phase was evaporated and heated in acetic anhydride and pyridine after which the product, the acetate derivative, was washed with dilute hydrochloric acid and water. The described method is both rapid and simple to apply, and produces a stable derivative. It should become widely used for measuring peptidoglycan in chemically complex environments.

Bacterial peptidoglycan polysaccharides in sterile human spleen induce proinflammatory cytokine production by human blood cells.

Peptidoglycan (PG) is the major component of the cell wall of gram-positive bacteria. In vitro, PG isolated from conventional bacterial cultures can induce secretion of proinflammatory cytokines by human monocytes, indicating that PG may be involved in immune responses against infections by gram-positive bacteria. To investigate the biologic activity of PG in human tissues, an improved method was developed to isolate significant amounts of PG from sterile human spleen tissue. Biochemical analysis demonstrated that PG isolated from human spleen is largely intact. Human whole blood cell cultures were able to produce the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 and -6 after stimulation with PG isolated from human spleen. Cytokine induction was not sensitive to inhibition by polymyxin B, in contrast to lipopolysaccharide. Collectively, the data show that intact PG in sterile human tissue is biologically active and may induce local proinflammatory cytokine production.

Total and viable airborne bacterial load in two different agricultural environments using gas chromatography-tandem mass spectrometry and culture: a prototype study.

Airborne exposure to bacterial components found in agricultural environments can lead to pulmonary inflammation. Total (viable and nonviable) bacterial load was monitored in a stable and a dairy by a new approach, gas chromatography-tandem mass spectrometry measurement of muramic acid, a component of gram positive and gram negative bacterial peptidoglycan. Also used to assess the gram negative bacterial load were 3-hydroxy fatty acids, markers of bacterial lipopolysaccharide. Culture, an established procedure for assessing the viable bacterial portion of airborne dust, served as a basis for comparison. The muramic acid and 3-hydroxy fatty acid concentrations (total C12:0, C14:0, and C16:0) showed a correlation with an R2 of 0.81. Dust and muramic acid levels also correlated. However, although relative muramic acid levels were lower in the stable than the dairy, colony forming units (CFU) were considerably higher in the stable. The total bacterial load (estimated from muramic acid values) for both the stable and dairy was also higher than would have been predicted from culture. These results suggest that nonculture based approaches and culture provide complementary but independent measurements of airborne biopollution.

On the origin of membrane vesicles in gram-negative bacteria.

It is proposed that the genesis of extracellular membrane vesicles in Gram-negative bacteria is a result of cell wall turnover. Peptidoglycan turnover would cause a turgor on the outer membrane, causing the outer membrane to bulge and finally bleb. Mechanical motion would then shear the blebs into the culture medium.

Inhalation of swine dust induces cytokine release in the upper and lower airways.

In healthy subjects, acute inhalation of swine dust causes an influx of inflammatory cells into the airways and increased bronchial responsiveness. The exposure may also cause fever and generalized symptoms. It seems likely that proinflammatory cytokines are involved in the response to inhaled swine dust. Nasal and bronchoalveolar lavage (BAL) were performed before, and 7 and 24 h after the start of 3 h exposure to swine dust, during a period of work in a swine confinement building, in 22 healthy subjects. Lavage fluids were analysed with regard to the cellular response and concentrations of interleukin (IL)-1 alpha, IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha). Each subject carried personal samplers for exposure measurements. Inhalable dust and airborne endotoxin, 3-hydroxylated (2-OH) fatty acid and muramic acid were measured. Bronchial responsiveness to methacholine was investigated 1-2 weeks before and 7 h after the start of the exposure. Exposure caused fever (> 38 degrees C) in three subjects, and approximately 25% of the subjects experienced symptoms. Bronchial responsiveness to methacholine increased by 3.5 (1.6-4.8) doubling doses (median (25th-75th percentile)). Following exposure, granulocytes increased more than 50 fold in BAL fluid and more than 40 fold in nasal lavage fluid. IL-1 alpha and IL-1 beta increased significantly in BAL fluid (p < 0.05) and nasal lavage fluid (p < 0.01). IL-6 increased 25 fold in BAL and 15 fold in nasal lavage fluid (p < 0.001). TNF-alpha was below detection limit (0.25 ng.L-1) in most subjects before exposure and increased following exposure to 3.8 (2.4-5.7) and 1.3 (0.6-2.3) ng.L-1 in BAL and nasal lavage fluid, respectively, (p < 0.001). Total inhalable dust was 20.5 (14.6-30.0) mg.m-3 and the concentrations of airborne endotoxin, 3-OH fatty acid and muramic acid were 1.2 (0.8-1.4), 3.5 (2.2-4.5) and 0.9 (0.3-1.9) microgram.m-3, respectively. There was a significant correlation between the IL-6 response in BAL fluid and exposure to dust endotoxin activity and 3-OH fatty acids (p < 0.05). Otherwise, no significant correlations were found between exposure and the cytokine response. We conclude that exposure to swine dust causes an intense upper and lower airway inflammation, which involves the proinflammatory cytokines interleukin-1, interleukin-6 and tumour necrosis factor-alpha.